Membrane-penetrating peptides to enhance transfection and compositions and methods for using same

ABSTRACT

The present invention is directed to non-naturally occurring peptides containing a membrane-penetrating amino acid sequence and further at least one polycationic moiety or peptide sequence. The peptides are suitable for use in delivery a cargo to the interior of a cell. Suitable cargo includes nucleic acid molecules (including DNA, RNA or PNA), polypeptides, or other biologically active molecules. The present invention is further directed to transfection complexes containing the non-naturally occurring peptides of the present invention in non-covalent association with at least one cationic lipid and a cargo to be delivered to the interior of a cell. The invention further relates to methods for the preparation and use of the non-naturally occurring peptides for the formation of transfection complexes and the delivery of a cargo to the interior of a cell in culture, an animal or a human. The invention also relates to compositions and kits useful for transfecting cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims the right of priorityunder 35 U.S.C. § 121 to U.S. application Ser. No. 14/569,583, filedDec. 12, 2014, now U.S. Pat. No. 9,856,496 B2, which claims the right ofpriority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser.No. 61/915,429, filed Dec. 12, 2013. The aforementioned applications arecommonly owned with the present application and the entire contentsthereof are hereby expressly incorporated by reference in their entiretyas though fully set forth herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 3, 2015, isnamed LT00804_SL.txt and is 35,579 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to the fields of transfectionand cell culture. In particular, the invention provides peptides whichare suitable for use as a cell-penetrating peptide, transfectioncomplexes containing the peptides and use thereof for the intracellulardelivery of cargo.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BACKGROUND

Lipid aggregates such as liposomes have been found to be useful asdelivery agents to introduce macromolecules, such as DNA, RNA, proteins,and small chemical compounds such as small molecules or pharmaceuticallyactive molecules, to cells and tissues in laboratory and clinicalresearch settings. In particular, lipid aggregates comprising cationiclipid components have been shown to be especially effective fordelivering anionic molecules to cells. In part, the effectiveness ofcationic lipids, and positively charged complexes formed with cationiclipids, is thought to result from enhanced affinity for cells, many ofwhich bear a net negative charge. Also in part, the net positive chargeon lipid aggregates comprising a cationic lipid enables the aggregate tobind polyanions, such as nucleic acids. Lipid aggregates containing DNAand RNA are known to be effective agents for efficient transfection oftarget cells.

The structure of various types of lipid aggregates varies, depending onthe composition and method of forming the aggregate. Such aggregatesinclude liposomes, unilamellar vesicles, multilameller vesicles,micelles and the like, having particular sizes in the nanometer tomicrometer range. Methods of making lipid aggregates are generally knownin the art. The main drawback to use of conventional phospholipidcontaining liposomes for delivery is the liposome composition has a netnegative charge which is not attracted to the negatively charged cellsurface. By combining cationic lipid compounds with a phospholipid,positively charged vesicles and other types of lipid aggregates can bindnucleic acids, which are negatively charged, can be taken up by targetcells, and can transfect target cells. (Felgner, P. L. et al. (1987)Proc. Natl. Acad. Sci. USA 84:7413-7417; Eppstein, D. et al., U.S. Pat.No. 4,897,355.).

Methods for incorporating cationic lipids into lipid aggregates are wellknown in the art. Representative methods are disclosed by Felgner etal., supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. et al.(1965) M. Mol. Biol. 23:238-252; Olson, F. et al. (1979) Biochim.Biophys. Acta 557:9-23; Szoka, F. et al. (1978) Proc. Natl. Acad. Sci.USA 75:4194-4198; Mayhew, E. et al. (1984) Biochim. Biophys. Acta775:169-175; Kim, S. et al. (1983) Biochim. Biophys. Acta 728:339-348;and Fukunaga, M. et al. (1984) Endocrinol. 115:757-761. Commonly usedtechniques for preparing lipid aggregates of appropriate size for use asdelivery vehicles include sonication and freeze-thaw plus extrusion.See, e.g., Mayer, L. et al. (1986) Biochim. Biophys. Acta 858:161-168.Microfluidization is used when consistently small (50 nm to 200 nm) andrelatively uniform aggregates are desired (Mayhew, E., supra). Cationiclipids have also been used in the past to deliver interfering RNA (RNAi)molecules to cells (Yu et al. (2002) PNAS 99: 6047-6052; Harborth et al.(2001) Journal of Cell Science 114:4557-4565).

The use of cationic lipids has become increasingly popular since itsintroduction over 15 years ago. Several cationic lipids have beendescribed in the literature and some of these are commerciallyavailable. DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride) was the first cationic lipid to be synthesized for the purposeof nucleic acid transfection. See Felgner et al. (Proc. Nat'l Acad. Sci.84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA can be formulated aloneor can be combined with DOPE (dioleoylphosphatidylethanolamine) into aliposome, and such liposomes can be used to deliver plasmids into somecells. Other classes of lipids subsequently have been synthesized byvarious groups. For example, DOGS(5-carboxyspermylglycinedioctadecylamide) was the first polycationiclipid to be prepared (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982(1989); U.S. Pat. No. 5,171,678) and other polycationic lipids havesince been prepared. The lipid DOSPA(2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanami-nium)has been described as an effective delivery agent (U.S. Pat. No.5,334,761).

In other examples, cholesterol-based cationic lipids, such as DC-Chol(N,N-dimethyl-N-ethylcarboxamidocholesterol) have been prepared and usedfor transfection (Gao et al. Biochem. Biophys. Res. Comm. 179, 280(1991)). In another example 1,4-bis(3-N-oleylamino-propyl)piperazine wasprepared and combined with histone H1 to generate a delivery reagentthat was reported to be less toxic than other reagents (Wolf et al.BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335). Several reagentsare commercially available. Some examples include LIPOFECTIN®(DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE® (DOSPA:DOPE)(Invitrogen), LIPOFECTAMINE® 2000 (Invitrogen) FUGENE®, TRANSFECTAM®(DOGS), EFFECTENE®, and DC-Chol.

None of these reagents can be used universally for all cells. This isperhaps not surprising in light of the variation in composition of themembranes of different types of cells as well as the barriers that canrestrict entry of extracellular material into cells. Moreover, themechanism by which cationic lipids deliver nucleic acids into cells isnot clearly understood. The reagents are less efficient than viraldelivery methods and are toxic to cells, although the degree of toxicityvaries from reagent to reagent.

However, transfection agents, including cationic lipids, are notuniversally effective in all cell types. Effectiveness of transfectionof different cells depends on the particular transfection agentcomposition. In general, polycationic lipids are more efficient thanmonocationic lipids in transfecting eukaryotic cells. In many cases,cationic lipids alone are not effective or are only partially effectivefor transfection.

While the use of lipid aggregates to introduce exogenous compounds intocells (a process known in the art as “transfection”) has become aroutine procedure in many labs and has been adapted for use in a widevariety of cell types and lineages, it is estimated that approximately60% of the cells and cell lines that routinely use this techniqueresearch and clinical settings are considered difficult to transfect,meaning they typically exhibit less than 60% transfection efficiency.Cells defined as difficult to transfect include primary cells, such asstem cells, progenitor cells, neuronal cells and other cell typesderived from neural tissues, primary blood cells (“PBMC”), HUVEC, andthe like, as well as certain cell lines that, while established, aredifficult to efficiently transfect using commercially availabletransfection reagent. Examples of difficult to transfect cell linesinclude PC12, HepG2, 3T3, LNCaP, A549, Jukat, and PC3, among others.

Over the last several decades, a number of naturally occurring peptidescapable of promoting the translocation of materials into a cell bypassing through the cell membrane. These so-called “membrane-penetratingpeptides” (“MPPs”) or “cell-penetrating peptides (“CPPs”) have been usedto promote the transport proteins, nucleic acids, polymers, or otherfunctional molecules into cells.

Membrane/cell-penetrating peptides (CPPs) such as theantennapedia-derived penetratin (Derossi et al., J. Biol. Chem., 269,10444-10450, 1994) and the Tat peptide (Vives et al., J. Biol. Chem.,272, 16010-16017, 1997) have been used to deliver cargo molecules suchas peptides, proteins and oligonucleotides (Fischer et al., Bioconjug.Chem., 12, 825-841, 2001) into cells. Areas of application range frompurely cell biological to biomedical research (Dietz and Bahr, Mol.Cell., Neurosci, 27, 85-131, 2004). Initially, cellular uptake wasbelieved to occur by direct permeation of the plasma membrane(Prochiantz, Cuff. Opin. Cell Biol., 12, 400-406, 2000). In recentyears, evidence has been mounting to indicate that at least some CPPsincrease cellular uptake of cargo by promoting endocytosis (for areview, see Fotin-Mleczek et al., Curr. Pharm. Design, 11, 3613-3628,2005). Given these recent results, the specification of a peptide as aCPP/MPP therefore does not necessarily imply a specific cellular importmechanism, but rather refers to a function as a peptide that, whenassociated with a cargo molecule, either covalently or non-covalently,enhances the cellular uptake of the cargo molecule.

There exists a need for additional reagents that enhance the delivery ofcargo and macromolecules into cells by improving transfection efficiencyof all cell in both research and clinical settings, particularly cellsthat are considered “difficult to transfect” (i.e., those cells that areeither refractory to transfection or that exhibit substantially lowertransfection efficiency than standard transformed cell lines routinelyused in laboratory settings), yet are easy to use and prepare andleverage the wide array of cationic lipid-based transfection reagentsthat are currently available.

SUMMARY

The present invention provides novel non-naturally occurring peptideshaving a cell penetrating function and being capable of formingtransfection complexes with a cargo molecule and one or more cationiclipids.

Disclosed herein are compositions and methods that provide improvedefficiency for introducing macromolecules, such as nucleic acids, intocells in culture or in a tissue in vivo. Accordingly, certainembodiments provide herein a complex containing, in non-covalentassociation, a cargo molecule, such as a nucleic acid molecule, atransfection agent and a non-naturally occurring peptide, where thenon-naturally occurring peptide contains a membrane-penetrating peptidesequence. In certain aspects, the complexes contain a macromolecule tobe introduced into the cell, such as a peptide, a protein, or a nucleicacid.

In one aspect of the invention, the non-naturally occurring peptides ofthe present invention have the general structure:A-L-B, orB-L-A;

Where A is membrane penetrating peptide, L is either a bond or a linkerpeptide, and B is a cationic moiety or a cationic polypeptide. In somepreferred though non-limiting embodiments, A is a peptide sequenceselected from those set forth in Table 1, or a variant thereof retainingat least a portion of its ability to enhance transfection efficiency. Insome embodiments, the peptide sequence of A is between 5 to about 50amino acids, and A is characterized in that it improves delivery of amolecule into a cell by at least 50% or more.

In some embodiments, the peptide sequence of A is between 5 to about 50amino acids, and A is characterized in that it improves delivery of amolecule into a cell by at 75% or more.

In some embodiments, A is at least 75% identical to any one of thepeptides set forth in Table 1, or a variant thereof retaining at least aportion of its ability to enhance transfection efficiency, where A ischaracterized in that it improves delivery of a molecule into a cell byat least 10% or more. In some embodiments, A is selected from the listconsisting of any one of SEQ ID NO. 1 through SEQ ID NO. 68, or avariant thereof.

In one aspect of the invention, the non-naturally occurring peptide isselected from any one of the peptides set forth in Table 4.

Further embodiments of the present invention are directed totransfection complexes containing the non-naturally occurring peptidesdescribed above in combination with one or more transfection reagents,which transfection reagents may include one or more cationic lipids, andoptionally one or more helper and/or neutral lipids.

In some embodiments, a transfection complex may include a cargo to bedelivered to the interior of a cell, or optionally may be administeredto an animal or to a human patient who would benefit from theadministration thereof. In some exemplary though non-limitingembodiments, preferred cargo molecules suitable for use with the presentinvention include nucleic acid molecules such as DNA molecules or RNAmolecules. Suitable DNA molecules may include a DNA molecule having anexpressible nucleic acid sequence, such as an expression vector or acDNA molecule comprising an open reading frame encoding a protein. Othersuitable molecules that may function as suitable cargo in the practiceof the present invention include RNA molecules, such as an mRNA moleculeor an RNAi molecule.

Further embodiments of the present invention are directed to methods forpreparing transfection complexes and to methods for the use thereof todeliver a cargo molecule to the interior of a cell. Methods forpreparing a transfection complex can include contacting a cargo moleculewith at least one cationic lipid or transfection reagent and thenon-naturally occurring peptides of the present invention, optionally inthe presence of one or more helper lipids and/or one or more neutrallipids, under conditions that promote the formation of a transfectioncomplex capable of conveying the cargo to the interior of a cell.

Further embodiments of the present invention are directed to methods fortransfecting cells that include forming transfection complexescomprising at least one cargo molecule, at least one cationic lipid ortransfection reagent, and a non-naturally occurring peptides inaccordance with the present invention, optionally having one or morehelper lipids and/or one or more neutral lipids, and contacting thetransfection complex with a cell under conditions that promote thetransfection of the cell. Yet further embodiments of the presentinvention are directed to pharmaceutical preparations comprising a cargoor a drug to be delivered to an animal or a human subject, at leastcationic lipid or transfection reagent and a non-naturally occurringpeptide of the present invention, optionally in the presence of one ormore helper lipids and/or one or more neutral lipids, to form apharmaceutically active complex suitable for the delivery of a drug orbiologically active compound to an animal or to a human subject havingneed thereof for the treatment of a physiological condition or disorder.

Other embodiments of the present invention will be apparent to one ofordinary skill in light of the following drawings and description of theinvention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings in which:

FIG. 1A shows a panel of 10 different cancer cell lines expressing GreenFluorescent Protein (GFP) that were transfected with an expressionvector encoding GFP using LIPOFECTAMINE® 3000 in combination with apeptide according to one embodiment;

FIG. 1B shows 2 different neuronal cell lines expressing GFP that weretransfected with an expression vector encoding GFP using LIPOFECTAMINE®3000 in combination with a peptide according to one embodiment;

FIG. 1C shows 2 different myoblast cell lines expressing GFP that weretransfected with an expression vector encoding GFP using LIPOFECTAMINE®3000 in combination with a peptide according to one embodiment;

FIG. 1D shows a kidney fibroblast cell line expressing GFP that weretransfected with an expression vector encoding GFP using LIPOFECTAMINE®3000 in combination with a peptide according to one embodiment;

FIG. 2 shows a panel of six different cell lines expressing GFP thatwere transfected with an expression vector encoding GFP using a theindicated commercially available transfection reagent; FUGENE® HD (firstcolumn), LIPOFECTAMINE® 2000 (center column); and LIPOFECTAMINE® 3000 incombination with a peptide according to one embodiment (last column);

FIG. 3A is a graph comparing the relative transfection efficiency for anexpression vector encoding GFP transfected into cultured HeLa cellsusing increasing dosages of three different commercially available lipidaggregate formulations, LIPOFECTAMINE® 2000 (open circles),LIPOFECTAMINE® LTX (open squares), and LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (open triangles);

FIG. 3B is a graph comparing the intensity of GFP expression in HeLacells transfected with an expression vector encoding GFP usingincreasing dosage of three different commercially available lipidaggregate formulations, LIPOFECTAMINE® 2000 (open circles),LIPOFECTAMINE® LTX (open squares), and LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (open triangles);

FIG. 4 is a Western blot comparing the relative expression levels of aGST-STAT fusion protein (upper panel) in HepG2 cells transfected with anexpression vector encoding a GST-STAT fusion protein using the followingcommercially available lipid aggregate formulations: LIPOFECTAMINE® 2000(first lane), LIPOFECTAMINE® 3000 in combination with a peptideaccording to one embodiment (second lane), FUGENE® HD (third lane), andX-TREMEGENE™ HP (last lane). The bottom panel shows a western blot ofendogenous β-actin to confirm equal loading of cytosolic extract in eachlane;

FIG. 5A is a graph comparing relative transfection efficiency of the H9human embryonic stem cell line (37,500 cells per well of a 96 wellplate) transfected with increasing dose of a GFP expression vector (50μg, left panel; 100 μg center panel, and 200 μg right panel) and usingbetween 0.1 to 0.6 μl per well of either LIPOFECTAMINE® 2000 (opentriangles) or LIPOFECTAMINE® 3000 in combination with a peptideaccording to an embodiment;

FIG. 5B is a representative fluorescence image of GFP expression in H9cells cultured in 96 well plates transfected with 100 μg/well using 200μl of either LIPOFECTAMINE® 2000 (left panel, demonstrating 18%transfection efficiency of H9 cells) or LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (right panel);

FIG. 6A shows the genomic modification efficiency of U2OS cells using acommercially available system as measured by mean Orange Fluorescentprotein (OFP) intensity (bar graph, upper panel) and representativefluorescence images (lower panel) od OFP expression in modified U2OScells using the indicated about of LIPOFECTAMINE® 2000 or LIPOFECTAMINE®3000 in combination with a peptide according to an embodiment;

FIG. 6B shows the genomic modification efficiency of HepG2 cells using acommercially available system as measured by mean OFP intensity (bargraph, upper panel) and representative fluorescence images (lower panel)od OFP expression in modified HepG2 cells using the indicated about ofLIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 in combination with a peptideaccording to an embodiment;

FIG. 7A shows the cleavage efficiency for TALENs and CRISPRs targetingthe AAVS1 locus in U2OS cells using either LIPOFECTAMINE® 2000 orLIPOFECTAMINE® 3000 in combination with a peptide according to anembodiment;

FIG. 7B shows the cleavage efficiency for TALENs and CRISPRs targetingthe AAVS1 locus in HepG2 cells using either LIPOFECTAMINE® 2000 orLIPOFECTAMINE® 3000 in combination with a peptide according to anembodiment;

FIG. 8 are bar graphs depicting relative transfection efficiency (uppergraph, GFP+as % of Single Cells Only) or relative expression level percell (lower graph; Single cells Only Mean FL-1) of HeLa cellstransfected with a GFP expression vector using the indicated doses (inμl) of LIPOFECTAMINE® 3000 alone (LF3K), LIPOFECTAMINE® 2000 (LF2K), apeptide according to an embodiment (Peptide 1) or LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (LF3K+Peptide 1);

FIG. 9A is a depiction of a peptide map of various peptides or peptidefragments used in the experiments depicted in FIGS. 9B and 9C in HepG2cells, in which Peptide A is the MPP Peptide alone, Peptide B is theLinker peptide alone, Peptide C is the Cationic peptide alone, Peptide Dis the Linker peptide fused to the Cationic peptide, and peptide E is afull length peptide having Peptide A fused Peptide D;

FIG. 9B depicts a series of fluorescence images to detect GFP expressionin cultured HepG2 cells transfected with an expression vector encodingGFP using LIPOFECTAMINE® 3000 in the presence of the indicated peptideor combination of peptides (shown in FIG. 9A);

FIG. 9C depicts two bar graphs showing mean fluorescence per cell (uppergraph) and transfection efficiency (% GFP+cells) in HepG2 cellstransfected with an expression vector encoding GFP using LIPOFECTAMINE®3000 in the presence of one of the indicated peptides A-E or theindicated combination of peptides (shown in FIG. 9A);

FIG. 10A is a depiction of a peptide map of various peptides or peptidefragments used in the experiments depicted in FIGS. 10B and 10C in A549cells, in which Peptide A is the MPP Peptide alone, Peptide B is theLinker peptide alone, Peptide C is the Cationic peptide alone, Peptide Dis the Linker peptide fused to the Cationic peptide, and peptide E is afull length peptide having Peptide A fused Peptide D;

FIG. 10B depicts a series of fluorescence images to detect GFPexpression in cultured A549 cells transfected with an expression vectorencoding GFP transfected with LIPOFECTAMINE® 3000 in the presence of theindicated peptide or combination of peptides (shown in FIG. 10A);

FIG. 10C depicts two bar graphs showing mean fluorescence per cell(upper graph) and transfection efficiency (% GFP+cells) in A549 cellstransfected with an expression vector encoding GFP using LIPOFECTAMINE®3000 in the presence of one of the indicated peptides A-E or theindicated combination of peptides (shown in FIG. 10A);

FIG. 11A is a depiction of a peptide map of various peptides or peptidefragments used in the experiments depicted in FIGS. 11B and 11C inMDA-MB-231 cells, in which Peptide A is the MPP Peptide alone, Peptide Bis the Linker peptide alone, Peptide C is the Cationic peptide alone,Peptide D is the Linker peptide fused to the Cationic peptide, andpeptide E is a full length peptide having Peptide A fused Peptide D;

FIG. 11B depicts a series of fluorescence images to detect GFPexpression in cultured MDA-MB-231 cells transfected with an expressionvector encoding GFP transfected with LIPOFECTAMINE® 3000 in the presenceof the indicated peptide or combination of peptides (shown in FIG. 11A);

FIG. 11C depicts two bar graphs showing mean fluorescence per cell(upper graph) and transfection efficiency (% GFP+cells) in MDA-MB-231cells transfected with an expression vector encoding GFP usingLIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A-Eor the indicated combination of peptides (shown in FIG. 11A);

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides improved reagents and compositions thatare suitable for the transfection of cells. In particular, the presentinvention provides compositions and reagents that enhance thetransfection efficiency of all cells, including those cell types thatare considered to typically be difficult to transfect. The compositionsand reagents of the present invention, when used in accordance with themethods described herein as well as with the general knowledge andexpertise within the purview of one having ordinary skill level in theart can typically increase the transfection efficiency of such by up to25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%,up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to90%, up to 95%, up to 100% or in excess of 100%. The inventionaccomplishes this by providing novel peptides comprising a cell/membranepenetrating peptide sequence used in combination with one or moretransfection lipids as described in greater detail below.

Definitions

The terms used throughout this specification generally have theirordinary meanings in the art, within the context of the invention, andin the specific context where each term is used. Certain terms arediscussed below, or elsewhere in the specification, to provideadditional guidance to the practitioner in describing the variousembodiments of the invention and how to make and use them. It will beappreciated that the same concept can be expressed in more than one way.Consequently, alternative language and synonyms may be used for any oneor more of the terms discussed herein, nor is any special significanceto be placed upon whether or not a term is elaborated or discussed ingreater detail herein. Synonyms for certain terms may be provided. Arecital of one or more synonyms does not exclude the use of othersynonyms. The use of examples anywhere in this specification, includingexamples of any terms discussed herein, is illustrative only, and in noway limits the scope and meaning of the invention or of any exemplifiedterm.

The term “introduction” when used in the context of introducing amacromolecule into cell culture refers to the provision of themacromolecule or compound into the culture medium with the understandingthat the goal of introducing the macromolecule is to enable the transferof macromolecule from the extracellular compartment to the cytoplasmiccompartment of the cultured cell.

The term “introduction” of a macromolecule or compound into at least onecell refers to the provision of a macromolecule or compound to a cell,such that the macromolecule or compound becomes internalized in thecell. For example, a macromolecule or compound can be introduced into acell using transfection, transformation, injection, and/or liposomalintroduction, and may also be introduced into a cell using other methodsknown to those of ordinary skill in the art. Preferably, a macromoleculeor compound is introduced into a cell by liposomal introduction. Themacromolecule is preferably a protein, peptide, polypeptide, or nucleicacid. The macromolecule may a protein. Alternatively, the macromoleculemay be a peptide. Alternatively, the macromolecule may be a polypeptide.The macromolecule may also be a nucleic acid.

The term “cargo”, when used herein in the context of the delivery of acargo into the interior of a cell, such as by mean of transfection,generally refers to any substance that is to be conveyed to the interiorof a cell, either in culture in a laboratory or in a tissue in an animalor a human. A cargo may, depending on the application, be amacromolecule such as a nucleic acid, a protein, or a peptide, or may bea drug or other organic small molecule.

The term “macromolecule,” as used herein, encompasses biomolecules. Inone embodiment, the term macromolecule refers to nucleic acid. In apreferred embodiment, the term macromolecule refers to deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). In some embodiments, the termmacromolecule refers to DNA. The DNA can be either linear DNA orcircular DNA, such as DNA in the form of a circular plasmid, an episomeor an expression vector. In certain preferred though non-limitingembodiments, the term macromolecule refers to complementary DNA (cDNA)have an expressible nucleic acid sequence, including at least one openreading frame operably linked to one or more nucleic acid sequencerequired for the transcription of an mRNA from the expressible nucleicacid sequence. A macromolecule can be charged or uncharged. A DNAmolecule is an example of a charged macromolecule. In some instances,the term “macromolecule”, as used herein, may be used interchangeablywith the terms “expressible nucleic acid” and “expression vector”. Inother embodiments, the term “macromolecule refers to an RNA molecule.The RNA molecule may be any type of RNA molecule, including but notlimited to an mRNA, a siRNA, a miRNA, an antisense RNA, a ribozyme, orany other type or species of RNA molecule familiar to those skilled inthe art without limitation, which would be sought to be delivered to theinterior of a cell.

The term “transfection” is used herein to mean the delivery of nucleicacid, protein or other macromolecule to a target cell, such that thenucleic acid, protein or other macromolecule is expressed or has abiological function in the cell.

The term “expressible nucleic acid” as used herein includes both DNA andRNA without regard to molecular weight, and the term “expression” meansany manifestation of the functional presence of the nucleic acid withinthe cell including, without limitation, both transient expression andstable expression. Functional aspects include inhibition of expressionby oligonucleotides or protein delivery.

The term “expression of nucleic acid” and their equivalents refer to thereplication of the nucleic acid in a cell, to transcription of DNA tomessenger RNA, to translation of RNA to protein, to post-translationalmodification of protein, and/or to protein trafficking in the cell, orvariations or combinations thereof.

The term “cell” as used herein refers includes all types of eukaryoticand prokaryotic cells. In preferred embodiments, the term refers toeukaryotic cells, especially cells grown in culture, or cells found in atissue in an animal or a human. In preferred embodiments, a cell refersto a mammalian cell. In certain exemplary though non-limitingembodiments, the term “cell” is meant to refer to any cell and cell linethat is routinely used in research and clinical settings, and mayinclude immortalized cell lines, transformed cell lines, or primarycells, without limitation.

The phrase “difficult to transfect”, or similar variants of the phrase,when used in the context of transfection procedures and reagents, is arelative term that generally refers to any cell or cell line thattypically exhibits less than 60% transfection efficiency whentransfected using standard commercially available transfection reagentssuch as, e.g., cationic lipids (examples of which include, but are notlimited to, LIPOFECTAMINE® 2000, LIPOFECTAMINE® LTX, LIPOFECTAMINE®,LIPOFECTIN®, FUGENE® HD, X-TREMEGENE™ HP, and the like). Cells typicallythought of as “difficult to transfect” include primary cells, such asstem cells, progenitor cells, neuronal cells and other cell typesderived from neural tissues, primary blood cells (“PBMC”), HUVEC, andthe like, as well as certain cell lines that, while established, aredifficult to efficiently transfect using commercially availabletransfection reagents. Examples of difficult to transfect cell linesinclude, but are not limited to, PC12, HepG2, 3T3, LNCaP, A549, Jurkat,primary cells, H9 embryonic stem cells, cultured embryonic stem cells,culture induced pluripotent stem cells (iPS cells), K-562, L6, L929,MCF-7, RAW 264.7, HT29, U937, Vero, HCT116, C6, C2C12, HL60, THP1, BHK,PC3, P19, SH-SY5Y, U2OS, HUH7, and PC3, among others. The recitationherein of various specific cell types and cell lines that are thought ofin the art is in no way meant to limit the scope of the presentinvention solely to those cell lines or close derivatives thereof, butis merely meant to illustrate the preponderance of cell lines and typescommonly used in laboratory settings that typically display less than60% transfection efficiency using commonly available cationic lipidbased transfections reagents, and which would benefit from the novelcompositions and formulations described herein to improve the relativetransfection efficiency by at least 5% or more.

By “cell culture” or “culture” is meant the maintenance of cells in anartificial, in vitro environment.

“Recombinant protein” refers to protein that is encoded by a nucleicacid that is introduced into a host cell. The host cell expresses thenucleic acid. The term “expressing a nucleic acid” is synonymous with“expressing a protein from an RNA encoded by a nucleic acid. “Protein”as used herein generically refers to any naturally occurring orsynthetic polymer of amino acids, e.g., peptides, polypeptides,proteins, lipoproteins, glycoproteins, etc.

As used herein, the term “polypeptide” generally refers to a naturallyoccurring, recombinant or synthetic polymer of amino acids, regardlessof length or post-translational modification (e.g., cleavage,phosphorylation, glycosylation, acetylation, methylation, isomerization,reduction, farnesylation, etc . . . ), that are covalently coupled toeach other by sequential peptide bonds. Although a “large” polypeptideis typically referred to in the art as a “protein” the terms“polypeptide” and “protein” are often used interchangeably. In general,the first amino acid residue or group of amino acid residues in apolypeptide are said to be at the “amino-terminal” or “N-terminal” ofthe polypeptide. Similarly, the last amino acid residue, or group ofamino acid residues in a polypeptide are said to be at the“carboxy-terminal” or “C-terminal”.

The term “peptide” as used herein is intended to be a generic term whichbroadly includes short peptides (typically less than 100 amino acids),polypeptides (typically more than 100 amino acids, and proteins (whichcontain one or more polypeptide chains). The peptides of this inventiontypically have more than two amino acids; preferred peptides have morethan 4 amino acids.

When used herein in the context of polypeptides described herein, theterms “variant”, “variants”, and the like, generally refer topolypeptide(s) that are structurally similar to a reference polypeptidebut are characterized by differences in amino acid sequence between thepolypeptides and the reference polypeptide (e.g., having at least 10%,at least 20%, at least 30%, at least 50%, at least 75%, at least 85%, orat least 95% sequence identity) and/or in the presence or absence of oneor more biochemical modifications (e.g., post-translationalmodifications, substitutions, adduct additions, and the like). While asubset of the general activities of certain variants may be similar,structural differences occurring between the variants may result in atleast a portion of their activities being non-overlapping. A “variant”may refer to a polypeptide molecule that is altered at one or morelocations in the polypeptide sequence, including additions, deletion,substitutions of one or more than one contiguous amino acid in thesequence, as well as covalent modifications of the molecule, relative tothe polypeptide molecule. Thus, in some instances, the terms “variant”and “isoform” may be used interchangeably. Illustrative examples of suchvariants would include, by way of example only, polypeptides in whichreplacement of a hydrogen group by an alkyl, acyl, thiol, amide or othersuch functional group has occurred at one or more amino acid residues. Avariant may have “conservative” changes, wherein a substituted aminoacid may have similar structural and/or chemical properties (e.g.,replacement of a non-polar amino acid residue with a different non-polaramino acid residue). A variant may also have “nonconservative” changes(e.g., replacement of a polar amino acid residue with a non-polar or acharged amino acid residue). Variants may also include similar minorvariations in amino acid sequence including, but not limited to,deletions, truncation, insertions, or combinations thereof. Guidance indetermining which amino acid residues may be substituted, inserted, ordeleted without abolishing or otherwise substantially affectingbiological activity is widely available in the art. Further guidance maybe found using computer programs well known in the art, for example,DNASTAR software. In general and in the context of the presentinvention, a variant will retain at least a subset of the biologicalfunctions typically associated with a known membrane penetratingpeptide, such as, for example, the ability to facilitate thetranslocation of a cargo colecule, such as, e.g., a nucleic acidmolecule, across a cell membrane to the cytosolic compartment thereof.

As used herein, the term “amino acid” generally refers to naturallyoccurring or synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to naturallyoccurring amino acids. Naturally occurring amino acids are those encodedby the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, carboxyglutamate, and O-phosphoserine.Amino acid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an α-carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine, andmethyl sulfonium. Such analogs have modified R groups (e.g., norleucineor norvaline) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. Amino acidmimetics refer to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunction in a manner similar to a naturally occurring amino acid. Theterm “amino acid” can refer to amino acids or their derivatives (e.g.,amino acid analogs), as well as their D- and L-forms. Examples of suchamino acids include glycine, L-alanine, L-asparagine, L-cysteine,L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine,L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine,L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine, N-acetyl cysteine.

“Kit” refers to transfection, DNA, RNAi, or other cargo (e.g., proteinor anionic molecule) delivery or protein expression or knockdown kitswhich include one or more of the reagents of the present invention ormixtures thereof. The kits may include one or more of the non-naturallyoccurring peptides described herein, optionally with one or morecationic lipids or transfections reagents. In some embodiments, thepeptide and the lipid reagents may be provided in a single formulation.In other embodiments, the lipid and the peptide may be providedseparately, with instruction to the user to combine the reagents at thetime of use. Such kits may comprise a carrying means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, test tubes and the like. Each of such containermeans comprises components or a mixture of components needed to performtransfection. Such kits may optionally include one or more componentsselected from any cargo molecules such as, e.g., nucleic acids(preferably one or more expression vectors, DNA molecules, RNA moleculesor RNAi molecules), cells, one or more compounds of the presentinvention, lipid-aggregate forming compounds, transfection enhancers,biologically active substances, etc.

The medium, methods, kit and composition of the present invention aresuitable for either monolayer or suspension culture, transfection, andcultivation of cells, and for expression of protein in cells inmonolayer or suspension culture. Preferably, the medium, methods, kitand composition of the present invention are for suspension culture,transfection, and cultivation of cells, and for expression of proteinproduct in cells in suspension culture.

By “culture vessel” is meant any container, for example, a glass,plastic, or metal container, that can provide an aseptic environment forculturing cells.

The term “combining” refers to the mixing or admixing of ingredients.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. Certain vectors usedin accordance with the practice of invention described herein may bewell-known vectors used in the art, such as, e.g., pCDNA 3.3, or amodified version thereof. Non-limiting examples of the types ofmodification to a vector that may be suitable in the practice of thepresent invention include, though are not limited to, modification suchas the addition of modification of one or more enhancers, one or morepromoters, one or more ribosomal binding sites, one or more origins ofreplication, or the like. In certain preferred though non-limitingembodiments, and expression vector used in the practice of the presentinvention may include one or more enhancer elements selected to improveexpression of the protein of interest in the present transientexpression system. The selected enhancer element may be positioned 5′ or3′ to the expressible nucleic acid sequence used to express the proteinof interest.

As used herein, the phrase “expression vector containing an expressiblenucleic acid” generally refers to a vector as defined above which iscapable to accommodating an expressible nucleic acid sequence having atleast one open-reading frame of a desired protein of interest (saidprotein of interest being selected by the user of the present invention)in additional to one or more nucleic acid sequences or elements that arerequired to support the expression thereof in a cell or in a cell-freeexpression system. Such additional nucleic acid sequences or elementsthat may be present in an expression vector as defined herein mayinclude, one or more promoter sequences, one or more enhancer elements,one or more ribosomal binding sites, one or more translationalinitiation sequences, one or more origins of replication, or one or moreselectable markers. A variety of nucleic acid sequences or elementsserving this purpose are familiar to the skilled artisan, and theselection of one or more thereof for use in the practice of the presentinvention is well within the purview of the skilled practitioner.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and refer to any nucleic acid, including deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). In preferred embodiments, “nucleicacid” refers to DNA, including genomic DNA, complementary DNA (cDNA),and oligonucleotides, including oligo DNA. In certain preferred thoughnon-limiting embodiments, “nucleic acid’ refers to genomic DNA and/orcDNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase or by asynthetic reaction. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and their analogs. If present,modification to the nucleotide structure may be imparted before or afterassembly of the polymer. The sequence of nucleotides may be interruptedby non-nucleotide components. A polynucleotide may comprisemodification(s) made after synthesis, such as conjugation to a label.Other types of modifications include, for example, “caps,” substitutionof one or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotides(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid or semi-solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,α-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs, and basic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO, or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

As used herein, the term “RNA-interference” or “RNAi” generally refersto the process of sequence-specific post-transcriptional gene silencing.RNAi is a process by which specific mRNAs are degraded into short RNAs.To mediate RNAi, a double-stranded RNA (dsRNA) with substantial sequenceidentity to the target mRNA is introduced into a cell. The target mRNAis then degraded in the cell, resulting in decreased levels of that mRNAand the protein it encodes.

As used herein, the term “RNAi construct” generally refers to smallinterfering RNAs (siRNAs), hairpin RNAs, and other RNA species that canbe cleaved in vivo to form siRNAs. The term also encompasses expressionvectors capable of giving rise to transcripts that form dsRNAs orhairpin RNAs in cells, and/or transcripts that can produce siRNAs invivo. The term “RNAi expression vector” refers to replicable nucleicacid constructs used to express (transcribe) RNA that produces siRNAduplexes in a host cell in which the construct is expressed.

As used herein, the term “short-interfering RNA” or “siRNA” generallyrefers to a short (approximately 19 to about 25 nucleotides in length),double stranded RNA molecule of defined nucleotide sequence that iscapable of mediating RNAi.

As used herein the terms “complexation reaction,” “complexation media”or the like, generally refer to a physiologically acceptable culturemedia or reaction in which a nucleic acid is complexed to a transfectionreagent formulation. Typically, a nucleic acid that is to be introducedinto a cell for the purpose of expressing a protein is first complexedwith a suitable transfection reagent (such as, e.g., a cationic lipidformulation) to lipid/nucleic acid complexes or aggregates.

Drug refers to any therapeutic or prophylactic agent other than foodwhich is used in the prevention, diagnosis, alleviation, treatment, orcure of disease in man or animal.

A variety of techniques and reagents are available for the introductionof macromolecules into a target cell in a process known as“transfection”. Commonly used reagents include, for example, calciumphosphate, DEAE-dextran and lipids. For examples of detailed protocolsfor the use of reagents of these types, numerous references texts areavailable for example, Current Protocols in Molecular Biology, Chapter9, Ausubel, et al. Eds., John Wiley and Sons, 1998. Additional methodsfor transfecting cells are known in the art, and may includeelectroporation (gene electrotransfer), sono-poration, opticaltransfection, protoplast fusion, impalefection, magnetofection, or viraltransduction.

A “reagent for the introduction of macromolecules” into cells or a“transfection reagent” is any material, formulation or composition knownto those of skill in the art that facilitates the entry of amacromolecule into a cell. For example, see U.S. Pat. No. 5,279,833. Insome embodiments, the reagent can be a “transfection reagent” and can beany compound and/or composition that increases the uptake of one or morenucleic acids into one or more target cells. A variety of transfectionreagents are known to those skilled in the art. Suitable transfectionreagents can include, but are not limited to, one or more compoundsand/or compositions comprising cationic polymers such aspolyethyleneimine (PEI), polymers of positively charged amino acids suchas polylysine and polyarginine, positively charged dendrimers andfractured dendrimers, cationic β-cyclodextrin containing polymers(CD-polymers), DEAE-dextran and the like. In some embodiments, a reagentfor the introduction of macromolecules into cells can comprise one ormore lipids which can be cationic lipids and/or neutral lipids.Preferred lipids include, but are not limited to,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamonium chloride (DOTMA),dioleoylphosphatidylcholine (DOPE),1,2-Bis(oleoyloxy)-3-(4′-trimethylammonio) propane (DOTAP),1,2-dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol (DOTB),1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl(4′-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide(CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),O,O′-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammoniumchloride, spermine conjugated to one or more lipids (for example,5-carboxyspermylglycine dioctadecylamide (DOGS),N,N^(I),N^(II),N^(III)-tetramethyl-N,N^(I),N^(II),N^(III)-tet-rapalmitylspermine(TM-TPS) and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde(DPPES)), lipopolylysine (polylysine conjugated to DOPE), TRIS(Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fatty acids(TFAs) and/or peptides such as trilysyl-alanyl-TRIS mono-, di-, andtri-palmitate, (3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-Chol), N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride(TMAG), dimethyl dioctadecylammonium bromide (DDAB),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-iniumtrifluoroacetate(DOSPA) and combinations thereof.

Those skilled in the art will appreciate that certain combinations ofthe above mentioned lipids have been shown to be particularly suited forthe introduction of nucleic acids into cells for example a 3:1 (w/w)combination of DOSPA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTAMINE™, a 1:1(w/w) combination of DOTMA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTIN®, a 1:1(M/M) combination of DMRIE and cholesterol is available from LifeTechnologies Corporation, Carlsbad, Calif. under the trade name DMRIE-Creagent, a 1:1.5 (M/M) combination of TM-TPS and DOPE is available fromLife Technologies Corporation, Carlsbad, Calif. under the trade nameCELLFECTIN® and a 1:2.5 (w/w) combination of DDAB and DOPE is availablefrom Life Technologies Corporation, Carlsbad, Calif. under the tradename LIPFECTACE®. In addition to the above-mentioned lipid combinations,other formulations comprising lipids in admixture with other compounds,in particular, in admixture with peptides and proteins comprisingnuclear localization sequences, are known to those skilled in the art.For example, see international application no. PCT/US99/26825, publishedas WO 00/27795, both of which are incorporated by reference herein.

Lipid aggregates such as liposomes have been found to be useful asagents for the delivery of macromolecules into cells. In particular,lipid aggregates comprising one or more cationic lipids have beendemonstrated to be extremely efficient at the delivery of anionicmacromolecules (for example, nucleic acids) into cells. One commonlyused cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).Liposomes comprising DOTMA alone or as a 1:1 mixture withdioleoylphosphatidylethanolamine (DOPE) have been used to introducenucleic acids into cells. A 1:1 mixture of DOTMA:DOPE is commerciallyavailable from Life Technologies Corporation, Carlsbad, Calif. under thetrade name of LIPOFECTIN™. Another cationic lipid that has been used tointroduce nucleic acids into cells is1,2-bis(oleoyl-oxy)-3-3-(trimethylammonia) propane (DOTAP). DOTAPdiffers from DOTMA in that the oleoyl moieties are linked to thepropylamine backbone via ether bonds in DOTAP whereas they are linkedvia ester bonds in DOTMA. DOTAP is believed to be more readily degradedby the target cells. A structurally related group of compounds whereinone of the methyl groups of the trimethylammonium moiety is replacedwith a hydroxyethyl group are similar in structure to the Rosenthalinhibitor (RI) of phospholipase A (see Rosenthal, et al., (1960) J.Biol. Chem. 233:2202-2206.). The RI has stearoyl esters linked to thepropylamine core. The dioleoyl analogs of RI are commonly abbreviatedDOR1-ether and DOR1-ester, depending upon the linkage of the lipidmoiety to the propylamine core. The hydroxyl group of the hydroxyethylmoiety can be further derivatized, for example, by esterification tocarboxyspermine.

Another class of compounds which has been used for the introduction ofmacromolecules into cells comprise a carboxyspermine moiety attached toa lipid (see, Behr, et al., (1989) Proceedings of the National Academyof Sciences, USA 86:6982-6986 and EPO 0 394 111). Examples of compoundsof this type include dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES) and 5-carboxyspermylglycinedioctadecylamide (DOGS). DOGS is commercially available from Promega,Madison, Wis. under the trade name of TRANSFECTAM™.

A cationic derivative of cholesterol(3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol, DC-Chol) hasbeen synthesized and formulated into liposomes with DOPE (see Gao, etal., (1991) BBRC 179(1):280-285.) and used to introduce DNA into cells.The liposomes thus formulated were reported to efficiently introduce DNAinto the cells with a low level of cellular toxicity. Lipopolylysine,formed by conjugating polylysine to DOPE (see Zhou, et al., (1991) BBA1065:8-14), has been reported to be effective at introducing nucleicacids into cells in the presence of serum.

Other types of cationic lipids that have been used to introduce nucleicacids into cells include highly packed polycationic ammonium, sulfoniumand phosphonium lipids such as those described in U.S. Pat. Nos.5,674,908 and 5,834,439, and international application no.PCT/US99/26825, published as WO 00/27795. One particularly preferredthough non-limiting transfection reagent for delivery of macromoleculesin accordance with the present invention is LIPOFECTAMINE 2000™ which isavailable from Life technologies (see U.S. international application no.PCT/US99/26825, published as WO 00/27795). Another preferred thoughnon-limiting transfection reagent suitable for delivery ofmacromolecules to a cell is EXPIFECTAMINE™. Other suitable transfectionreagents include LIOFECTAMINE™ RNAiMAX, LIPOFECTAMINE™ LTX,OLIGOFECTAMINE™, Cellfectin™ INVIVOFECTAMINE™, INVIVOFECTAMINE™ 2.0, andany of the lipid reagents or formulations disclosed in U.S. Patent Appl.Pub. No. 2012/0136073, by Yang et al. (incorporated herein by referencethereto). A variety of other transfection reagents are known to theskilled artisan and may be evaluated for the suitability thereof to thetransient transfection systems and methods described herein.

The present invention provides improved reagents and compositions thatare suitable for the transfection of cells. In particular, the presentinvention provides compositions and reagents that enhance thetransfection efficiency of all cells, including those cell types thatare considered to typically be difficult to transfect. The compositionsand reagents of the present invention, when used in accordance with themethods described herein as well as with the general knowledge andexpertise within the purview of one having ordinary skill level in theart can typically increase the transfection efficiency of such cells byup to 10%, up to 15%, 20%, up to 25%, up to 30%, up to 35%, up to 40%,up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100% or in excessof 100%. The invention accomplishes this by providing novel peptidescomprising a cell/membrane penetrating peptide sequence used incombination with one or more transfection lipids for the delivery of acargo molecule, in particular but not limited to a nucleic acid moleculesuch as a DNA molecule or an RNA molecule, to the interior orcytoplasmic compartment of a cell in culture or a cell or tissue invivo, in particular but not limited to a cell that in considered“difficult to transfect”, as described in greater detail below.

Membrane/Cell-Penetrating Peptides

The present invention is directed to non-naturally occurring, syntheticpeptides that are used in combination with transfection reagents, whichtransfections reagents may preferably include though are not limited to,lipid-based transfection reagents, particularly cationic lipid-basedtransfection reagents, the inclusion of which in a transfection compleximprove the transfection efficiency of cells in part by enhancing thetransport of a cargo molecule, e.g., a nucleic acid molecule or anyother suitable cargo molecule such as will be readily apparent to oneskilled in the art, across the cell membrane such that the cargomolecule is delivered to the cytosolic compartment of a cell in cultureor in a tissue in vivo.

Ideally, the non-naturally occurring peptides of the present inventionwill be used to form a multi-component complex with a lipid aggregatecomposition and the cargo molecule such that the complex enhances thedelivery of the cargo molecule to the cytosolic compartment of the cellor tissue.

In one aspect of the invention, non-naturally occurring peptides arecontacted with at least one transfection reagent and at least one cargomolecule to form a transfection complex comprising the transfectionreagent, the cargo, and the peptide, and being characterized byimproving the transfection efficiency (measured as improved transfer ofthe cargo to the interior of a cell in culture or in a tissue in vivo)of a complex compared to an identical transfection complex that lacksthe non-naturally occurring peptide.

The selection of what constitutes an optimal transfection reagent foruse with the present invention depends on the identity and nature of thecargo to be delivered, the identity and characteristics of the cells tobe transfected, wither the transfection is to take place in isolatedcells in culture or in a tissue in an animal or a human in vivo, and theidentity of the non-naturally occurring peptide. All thesecharacteristics are well-known to the practitioner having ordinary skilllevel in the art, and the selection of an optimal transfection reagentin the specific context of specific applications, as well as the way todetermine what constitutes of optimal concentrations and formulation ofthe components is readily apparent to such a person without undueexperimentation and without departing from the spirit and scope of theinvention.

In certain preferred though non-limiting embodiments, the transfectionreagent selected for use in the formation of a transfection complex inaccordance with the embodiments set forth herein may be a cationiclipid, in particular a cationic lipid capable of forming lipidaggregates.

In some embodiments, a transfection complex may include a lipidaggregate composition, the lipid aggregate composition comprising atleast one cationic lipid, optionally more than one cationic lipid,optionally in the presence of at least one helper lipid, contacted witha cargo molecule and a least one non-naturally occurring peptides havingthe general structure:A-L-B, orB-L-A;

Where A is membrane penetrating peptide (MPP), L is either a covalentbond linking A to B or a linker peptide, and where B is either acationic polypeptide, a cationic moiety, or a cationic peptidecovalently linked to a cationic moiety, where the non-naturallyoccurring peptide is characterized in that the presence of non-naturallyoccurring peptide as a component of a transfection complex increases thetransfection efficiency of the transfection complex is enhanced orimproved up to 10%, up to 15%, 20%, up to 25%, up to 30%, up to 35%, upto 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%,up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%,up to 500% or in excess of 500% above that of an identical transfectioncomplex that lacks the non-naturally occurring peptide.

A may be any peptide, without limitation and independent of themechanism by which the peptide carries out its function, that is knownor can be demonstrated to enhance or to promote the transfer of amolecule, such as a cargo molecule as defined above, in particular anucleic acid molecule such as a DNA or an RNA molecule, from anextracellular compartment, such as, e.g., a cell culture medium or aninterstitial or body fluid, across a cell membrane such that the cargomolecule is conveyed to the cytoplasmic compartment of the cell where itcan effect at least one measurable biological response or function. Thedetermination of what constitutes “enhancement” of transfer across acell membrane is well within the skill level of a practitioner havingordinary skill level in the art, and the identification of a suitablepeptide or variant of a known peptide that functions to enhance orpromote the transfer of a cargo molecule in such a manner is readilyapparent to such a person using a wide variety of known techniques.

In some non-limiting embodiments, the peptide sequence of A may bebetween about 5 to about 75 amino acids, between about 5 to about 60amino acids, between about 5 to about 50 amino acids, between about 5 toabout 40 amino acids, between about 5 to about 30 amino acids, betweenabout 5 to about 20 amino acids or between about 5 to about 15 aminoacids, between about 10 to about 75 amino acids, between about 10 toabout 60 amino acids, between about 10 to about 50 amino acids, betweenabout 10 to about 40 amino acids, between about 10 to about 30 aminoacids, between about 10 to about 20 amino acids, or between about 10 toabout 15 amino acids, and where A is characterized in that the presenceof non-naturally occurring peptide as a component of a transfectioncomplex enhances the transfection efficiency of the transfection complexup to 10%, up to 15%, 20%, up to 25%, up to 30%, up to 35%, up to 40%,up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 150%,up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 500%or in excess of 500% above that of an identical transfection complexthat lacks the non-naturally occurring peptide.

A variety of peptide sequences suitable for use as an MPP (i.e., regionA of the structure A-L-B or B-L-A shown above) in the non-naturallyoccurring peptides as described herein are known in the art, any ofwhich may be used in the practice of the present invention withoutlimitation. A representative though non-limiting set of peptides thatare known to function as an MPP is shown on Table 1.

In some non-limiting embodiments, A is a peptide comprising a peptidesequence selected from any one of SEQ ID NO. 1-68, or a variant thereofhaving at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% or at least95% sequence similarity to any one of SEQ ID NO: 1-68 and retaining atleast 50% at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90% or at least 95%, atleast 155%, greater that 100%, up to 115%, up to 120%, up to 130%, up to140%, up to 150%, up to 160%, up to 170%, up to 180% of the functionthereof to enhance the delivery of a cargo molecule to the interior of acell.

In some embodiments, L may be a covalent bond linking A and B.

In some embodiments, L may comprise a dipeptide of neutral (uncharged atphysiologic pH) amino acids in which optionally one of the two aminoacids in the dipeptide comprises at least one polar side chain. In anembodiment, L may comprise a dipeptide comprising at least one polarside chain or at least one hydrophobic side chain, wherein said polar orhydrophobic side chain preferably is not a bulky side chain. In anembodiment L may comprise a dipeptide comprising at least one glycine,at least one, valine, at least one alanine, at least one serine or atleast one threonine. In some embodiments, L may comprise a dipeptideselected from the list consisting of GG, AA, GA, AG, AS, AY, GS, GT, GV,AV, SV, TV, VG, VA, and VT.

In some embodiments, L may be linker peptide having between about 3 toabout 50, about 45, about 40, about 35, about 30, about 25, about 20,about 15, about 14, about 13, about 12, about 11, about 10, about 9,about 8, about 7, about 6, about 5, about 4 amino acids, where at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90% or greater than about 90% of the amino acids areneutral.

In some embodiments, L may be linker peptide having between about 3 toabout 50, about 5 to about 25, about 6 to about 20, about 8 to about 15amino acids, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, whereup to about 35% of the amino acids contain a neutral polar side chain,and/or at least 35% of the amino acids contain a hydrophobic side chain,where the polar and hydrophobic side chains are not bulky side chains.

In some embodiments, L may be linker peptide having between about 3 toabout 50, about 5 to about 25, about 6 to about 20, about 8 to about 15amino acids, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, whereup to about 35% of the amino acids are selected from serine, threonine,valine, isoleucine, and leucine.

In some embodiments, L may be linker peptide having between about 3 toabout 50, about 5 to about 25, about 6 to about 20, about 8 to about 15amino acids, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, whereat least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80% ormore of the amino acids are glycine or alanine.

In some embodiments, L may be linker peptide having the structure:

X_(m)—Y_(n)

_(p) or

Y_(n)—X_(m)

_(p),

where each X is independently neutral amino acid with a non-polar sidechain, where each Y is independently a neutral amino acid with a polarside chain, and where m is an integer from 3 to 10, where n is aninteger from 1 to 5, and where when L is not a bond, p is an integerfrom 1 to 20. In an embodiment, m>n. In some embodiments, m is 2 and nis 1, or m is 3 and n is 1 or 2. In some embodiments, each X isindependently glycine, alanine, valine, leucine or isoleucine. In someembodiments, each Y is independently serine or threonine.

A variety of peptide sequences suitable for use as a Linker (i.e.,region L of the structure A-L-B or B-L-A shown above) in thenon-naturally occurring peptides as described herein may be used in thepractice of the present invention, any of which may be used in thepractice of the present invention without limitation. A representativethough non-limiting set of Linker peptides that are contemplated for usewith the embodiments described herein are set forth in Table 2.

In some non-limiting embodiments, L is a peptide comprising a peptidesequence selected from any one of SEQ ID NO. 69-81, or a variant thereofhaving at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% or at least95% sequence similarity to any one of SEQ ID NO. 69-81 and retaining atleast 50% at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90% or at least 95%, atleast 155%, greater that 100%, up to 115%, up to 120%, up to 130%, up to140%, up to 150%, up to 160%, up to 170%, up to 180% of the functionthereof to enhance the delivery of a cargo molecule to the interior of acell.

In some embodiments, B may be either a cationic polypeptide, a cationicmoiety, or a cationic peptide covalently linked to a cationic moiety.Any cationic moiety known in the art to impart a cationic charge to amolecule, in particular a peptide, may be selected for use in thepresent invention, without limitation. Preferred though non-limitingexamples of cationic moieties suitable for use in the present inventioninclude polyamines, such as, for example, one or more of putrescine,cadaverine, spermine, spermidine. Additional cationic moieties mayinclude poly-L-Lysine.

In some embodiments, B may be a peptide having a peptide sequencebetween about 3 to about 50 amino acids, about 5 to about 25, about 6 toabout 20, about 8 to about 15 amino acids, or about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 amino acids, where at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the amino acids are positivelycharged at physiologic pH.

A variety of peptide sequences suitable for use as a Cationic region(i.e., region B of the structure A-L-B or B-L-A shown above) in thenon-naturally occurring peptides as described herein may be used in thepractice of the present invention, any of which may be used in thepractice of the present invention without limitation. A representativethough non-limiting set of Linker peptides that are contemplated for usewith the embodiments described herein are set forth in Table 3.

TABLE 1 Exemplary Membrane Penetrating Peptide (MPP) sequences NamePeptide Sequence SEQ ID NO: DPV10/6 SRRARRSPRESGKKRKRKR 1 DPV15bCGAYDLRRRERQSRLRRRERQSR 2 YM-3 GYGRKKRRGRRRTHRLP 3 Penetration IGCRH 4Tat(46-57) CYGRKKRRQRRR 5 LR11 RILQQLLFIHF 6 C45D18DTWAGVEAIIRILQQLLFIHFR 7 Lyp-1 CGNKRTRGC 8 Lyp-2 CAGRRSAYC 9(42-38)(9-1)Crot GSGKKGGKKHCQKY 10 (1-9)(38-42)Crot YKQCHKKGGKKGSG 11BMV GAG KMTRAQRRAAARRNRWTARGC 12 hPER1-PTD(830-845)NLS GRRHHCRSKAKRSRHH13 hLF1 KCFQWQRNMRKVRGPPVSCIKR 14 hLF2 KCFQWQRNVRKVRGPPVSCIKR 15 hLF3KCFQWQRNIRKVRGPPVSCIKR 16 hLF4 KCFQWQRNXRKVRGPPVSCIKR, whereby X 17 isnorvaline hLF5 KCFQWQRNLRKVRGPPVSCIKR 18 hLF6 KCFQWQRNXRKVRGPPVSCIKR,whereby X 19 is norleucine hLF7 CFQWQRNVRKVRGPPVSC 20 hLF8CFQWQRNIRKVRGPPVSC 21 hLF9 CFQWQRNXRKVRGPPVSC, whereby X is 22 norvalinehLF10 CFQWQRNLRKVRGPPVSC 23 hLF11 CFQWQRNXRKVRGPPVSC, whereby X is 24norleucine hLF12 FQWQRNVRKVRGPPVS 25 hLF13 FQWQRNIRKVRGPPVS 26 hLF14FQWQRNVRKVRGPPVS, whereby X is 27 norvaline hLF15 FQWQRNLRKVRGPPVS 28hLF16 FQWQRNXRKVRGPPVS, whereby X is 29 norleucine hLF17 FQWQRNVRKVR 30hLF18 FQWQRNIRKVR 31 hLF19 FQWQRNXRKVR, whereby X is norvaline 32 hLF20FQWQRNLRKVR 33 hLF21 FQWQRNXRKVR, whereby X is norleucine 34 hLF22CFQWQRNMRKVRGPPVSC 35 C45D18 DTWAGVEAIIRILQQLLFIHFRIGCRH 36 LR20RILQQLLFIHFRIGCRHSRI 37 LR17 RILQQLLFIHFRIGCRH 38 LR15 RILQQLLFIHFRIGC39 LR15DL RIFIHFRIGC 40 LR8DHF RIFIRIGC 41 LR8DHFRI RIFIGC 42 LR8DRIHFFIRIGC 43 Tat YGRKKKRRQRRR 44 ΔNTat RKKRRQRRR 45 Antp RQIKIWFQNRRMKWKK46 bLF PEWFKCRRWQWRMKKLGA 47 bLF2 KCRRWQWRMKKLGAPSITCVRR 48 bLF3CRRWQWRMKKLGAPSITC 49 LF1 FQWQRNMRKVRGPPVS 50 LF2 FQWQRNMRKVR 51 SynB1RGGRLSYSRRRFSTSTGR 52 Penetratin PTD RQIKWFQNRRMKWKK 53 PTD-4PIRRRKKLRRLK 54 PTD-5 RRQRRTSKLMKR 55 FHV Coat-(35-49) RRRRNRTRRNRRRVR56 BMV Gag-(7-25) KMTRAQRRAAARRNRWTAR 57 HTLV-II Rex-(4-16)TRRQRTRRARRNR 58 D-Tat GRKKRRQRRRPPQ 59 R9-Tat GRRRRRRRRRPPQ 60Transportan GWTLNSAGYLLGKINLKALAALAKKIL 61 MAP KLALKLALKLALALKLA 62 SBPMGLGLHLLVLAAALQGAWSQPKKKRKV 63 FBP GALFLGWLGAAGSTMGAWSQPKKKRKV 64 MPGGALFLGFLGAAGSTMGAWSQPKKKRKV 65 MPG^((ΔNLS)) GALFLGFLGAAGSTMGAWSQPKSKRKV66 Pep-1 KETWWETWWTEWSQPKKKRKV 67 Pep-2 KETWFETWFTEWSQPKKKRKV 68

TABLE 2 Exemplary Linker (L) peptide sequences Name Peptide Sequence SEQID NO: Linker 1 GGGSGGGSGGGS 69 Linker 2 GGSGGSGGSGGS 70 Linker 3GGGGGGGGGGGG 71 Linker 4 GGGSGGGSGGGSGGGS 72 Linker 5 GGGAGGGAGGGAGGGA73 Linker 6 GGGAGGGSGGGAGGGS 74 Linker 7 AAAAAAAAAAA 75 Linker 8AAASAAASAAAS 76 Linker 9 AAASAAASAAASAAAS 77 Linker 10 AAGSAAGSAAGS 78Linker 11 AGGSAGGSAGGS 79 Linker 12 GGGTGGGTGGGT 80 Linker 13AAATAAATAAAT 81

TABLE 3 Exemplary cationic polypeptide (CP) sequences CP1 RRRRRRRRRRR 82CP2 RRRRRRRRRRRRRRRR 83 CP3 RRRRRRRRRRRRRRRRRRRRRR 84 CP4 KKKKKKKKKKK 85CP5 RRRRHRRRRHRRRRH 86 CP6 RRRRKRRRRKRRRRK 87 CP7 KKKKRKKKKRKKKKR 88

Table 4 set forth various peptide sequences that can be used in thepractice of the present invention, though it will be understood by oneof ordinary skill level in the art that the list of peptide sequences inTable 4 is provided by way of example only, and is not meant to limitthe scope of the invention solely to those sequences explicitly spelled.On the contrary, it will be readily apparent to such a person that,based on the teachings set forth above with regard to the A, L and Bregions of the inventive peptides, a large number of peptides that arepotentially useful in the practice of the invention set forth in hereinis possible. Moreover, it is well within the purview of the skilledartisan to determine whether a given peptide sequence falls within thescope of the invention using standard techniques in the art, withoutrequiring undue experimentation. Moreover, it will be appreciated thatvarious variants of the peptide sequences appearing in Table 4 also fallwithin the scope of the invention, as long as such variants satisy thestructural and functional characteristics set forth above. Variants ofthe peptide sequences appearing in Table 4, or of any other candidatepeptides not explicitly recited in Table 4 but satisfying the structuraland functional requirements set forth above can include deletions,insertion, substitutions with naturally occurring or non-proteinogenicamino acids.

TABLE 4 Exemplary Non-Naturally Occurring Peptides SEQ ID Name PeptideSequence NO: Peptide 1 SRRARRSPRESGKKRKRKRGGGSGGGSG 89 GGSRRRRRRRRRRRPeptide 2 CGAYDLRRRERQSRLRRRERQSRGGGSG 90 GGSGGGSRRRRRRRRRRR Peptide 3GYGRKKRRGRRRTHRLPGGGSGGGSGGG 91 SRRRRRRRRRRR Peptide 4IGCRHGGGSGGGSGGGSRRRRRRRRRRR 92 Peptide 5 CGNKRTRGCGGGSGGGSGGGSRRRRRRR93 RRRR Peptide 6 CAGRRSAYCGGGSGGGSGGGSRRRRRRR 94 RRRR Peptide 7GSGKKGGKKHCQKYGGGSGGGSGGGSR 95 RRRRRRRRRR Peptide 8YKQCHKKGGKKGSGGGGSGGGSGGGSR 96 RRRRRRRRRR Peptide 9KMTRAQRRAAARRNRWTARGCGGGSGG 97 GSGGGSRRRRRRRRRRR Peptide 10RRHHCRSKAKRSRHHGGGSGGGSGGGSR 98 RRRRRRRRRR Peptide 11KCFQWQRNMRKVRGPPVSCIKRGGGSGG 99 GSGGGSRRRRRRRRRRR Peptide 12PEWFKCRRWQWRMKKLGAGGSGGSGGS 100 GGSKKKKKKKKKKK Peptide 13KCFQWQRNVRKVRGPPVSCIKRAAGSAA 102 GSAAGSKKKKRKKKKRKKKKR Peptide 14GRRHHCRSKAKRSRHHGGGSGGGSGGGS 103 RRRRRRRRRRR Peptide 15CGNKRTRGCGGGGGGGGG 104 RRRRKRRRRKRRRRK Peptide 16 KCRRWQWRMKKLGAPSITCVRR105 Peptide 17 CYGRKKRRQRRRRRRRRRRRRRRRRRRR 106 RRRRRR Peptide 18RQIKWFQNRRMKWKKAAASAAASAAAS 107 RRRKKKRRRKKK

In some embodiments, the peptide sequence of A is between 5 to about 50amino acids, and A is characterized in that it improves delivery of amolecule into a cell in the presence of a cationic lipid-basedtransfection reagent by at least 10% or more, at least 15% or more, atleast 20% or more, at least 25% or more, at least 30% or more, at least35% or more, at least 40% or more, at least 45% or more, at least 50% ormore, at least 55% or more, at least 60% or more, at least 65% or more,at least 70% or more, at least 75% or more, at least 80% or more, atleast 85% or more, at least 90% or more, at least 95% or more, at least100%, at least 200% or more, at least 250% or more, at least 300% ormore, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more.

In some embodiments, A is at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% similar to any one of the peptide sequences setforth in Table 1, and A is characterized in that it improves delivery ofa molecule into a cell by at least 10% or more, at least 15% or more, atleast 20% or more, at least 25% or more, at least 30% or more, at least35% or more, at least 40% or more, at least 45% or more, at least 50% ormore, at least 55% or more, at least 60% or more, at least 65% or more,at least 70% or more, at least 75% or more, at least 80% or more, atleast 85% or more, at least 90% or more, at least 95% or more, at least100%, at least 200% or more, at least 250% or more, at least 300% ormore, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more.

In some embodiments, A may comprise any one or more of the peptidesequences set forth in SEQ ID NO. 1-68, or a variant thereof being atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%similar thereto and having at least 10% or more, at least 15% or more,at least 20% or more, at least 25% or more, at least 30% or more, atleast 35% or more, at least 40% or more, at least 45% or more, at least50% or more, at least 55% or more, at least 60% or more, at least 65% ormore, at least 70% or more, at least 75% or more, at least 80% or more,at least 85% or more, at least 90% or more, at least 95% or more, atleast 100%, at least 200% or more, at least 250% or more, at least 300%or more, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more of the activity thereof.

In some embodiments, A may comprise any one or more of the peptidesequences set forth in SEQ ID NO. 14-35, or a variant thereof being atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%similar thereto and having at least 10% or more, at least 15% or more,at least 20% or more, at least 25% or more, at least 30% or more, atleast 35% or more, at least 40% or more, at least 45% or more, at least50% or more, at least 55% or more, at least 60% or more, at least 65% ormore, at least 70% or more, at least 75% or more, at least 80% or more,at least 85% or more, at least 90% or more, at least 95% or more, atleast 100%, at least 200% or more, at least 250% or more, at least 300%or more, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more of the activity thereof.

In some embodiments, A may comprise any one or more of the peptidesequences set forth in SEQ ID NO. 37-43, or a variant thereof being atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%similar thereto and having at least 10% or more, at least 15% or more,at least 20% or more, at least 25% or more, at least 30% or more, atleast 35% or more, at least 40% or more, at least 45% or more, at least50% or more, at least 55% or more, at least 60% or more, at least 65% ormore, at least 70% or more, at least 75% or more, at least 80% or more,at least 85% or more, at least 90% or more, at least 95% or more, atleast 100%, at least 200% or more, at least 250% or more, at least 300%or more, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more of the activity thereof.

In some embodiments, A may comprise any one or more of the peptidesequences set forth in SEQ ID NO. 44, 45, 46, or a variant thereof beingat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%similar thereto and having at least 10% or more, at least 15% or more,at least 20% or more, at least 25% or more, at least 30% or more, atleast 35% or more, at least 40% or more, at least 45% or more, at least50% or more, at least 55% or more, at least 60% or more, at least 65% ormore, at least 70% or more, at least 75% or more, at least 80% or more,at least 85% or more, at least 90% or more, at least 95% or more, atleast 100%, at least 200% or more, at least 250% or more, at least 300%or more, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more of the activity thereof.

In some embodiments, A may comprise any one or more of the peptidesequences set forth in SEQ ID NO. 52-68, or a variant thereof being atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%similar thereto and having at least 10% or more, at least 15% or more,at least 20% or more, at least 25% or more, at least 30% or more, atleast 35% or more, at least 40% or more, at least 45% or more, at least50% or more, at least 55% or more, at least 60% or more, at least 65% ormore, at least 70% or more, at least 75% or more, at least 80% or more,at least 85% or more, at least 90% or more, at least 95% or more, atleast 100%, at least 200% or more, at least 250% or more, at least 300%or more, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more of the activity thereof.

In some embodiments, A may comprise any one or more of the peptidesequences set forth in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, SEQ.ID. NO. 5, SEQ ID NO. 13, and SEQ ID NO. 14, or a variant thereof beingat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%similar thereto and having at least 10% or more, at least 15% or more,at least 20% or more, at least 25% or more, at least 30% or more, atleast 35% or more, at least 40% or more, at least 45% or more, at least50% or more, at least 55% or more, at least 60% or more, at least 65% ormore, at least 70% or more, at least 75% or more, at least 80% or more,at least 85% or more, at least 90% or more, at least 95% or more, atleast 100%, at least 200% or more, at least 250% or more, at least 300%or more, at least 350% or more, at least 400% or more, at least 500% ormore, at least 600% or more, at least 700% or more, at least 800% ormore, at least 900% or more, at least 1000% or more at least 1500% ormore or at least 2000% or more of the activity thereof.

In one aspect of the invention, the non-naturally occurring peptide Amay comprise any one or more of the peptide sequences set forth in Table4, or a variant thereof being at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% similar thereto and having at least 10% or more,at least 15% or more, at least 20% or more, at least 25% or more, atleast 30% or more, at least 35% or more, at least 40% or more, at least45% or more, at least 50% or more, at least 55% or more, at least 60% ormore, at least 65% or more, at least 70% or more, at least 75% or more,at least 80% or more, at least 85% or more, at least 90% or more, atleast 95% or more, at least 100%, at least 200% or more, at least 250%or more, at least 300% or more, at least 350% or more, at least 400% ormore, at least 500% or more, at least 600% or more, at least 700% ormore, at least 800% or more, at least 900% or more, at least 1000% ormore at least 1500% or more or at least 2000% or more of the activitythereof.

In one aspect of the invention, the non-naturally occurring peptide Amay comprise any one or more of the peptide sequences set forth in SEQ.ID. NO. 89-107, or a variant thereof being at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% similar thereto and having atleast 10% or more, at least 15% or more, at least 20% or more, at least25% or more, at least 30% or more, at least 35% or more, at least 40% ormore, at least 45% or more, at least 50% or more, at least 55% or more,at least 60% or more, at least 65% or more, at least 70% or more, atleast 75% or more, at least 80% or more, at least 85% or more, at least90% or more, at least 95% or more, at least 100%, at least 200% or more,at least 250% or more, at least 300% or more, at least 350% or more, atleast 400% or more, at least 500% or more, at least 600% or more, atleast 700% or more, at least 800% or more, at least 900% or more, atleast 1000% or more at least 1500% or more or at least 2000% or more ofthe activity thereof.

In one aspect of the invention, the non-naturally occurring peptide Amay comprise any one or more of the peptide sequences set forth in SEQ.ID. NO. 89-96, or a variant thereof being at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% similar thereto and having atleast 10% or more, at least 15% or more, at least 20% or more, at least25% or more, at least 30% or more, at least 35% or more, at least 40% ormore, at least 45% or more, at least 50% or more, at least 55% or more,at least 60% or more, at least 65% or more, at least 70% or more, atleast 75% or more, at least 80% or more, at least 85% or more, at least90% or more, at least 95% or more, at least 100%, at least 200% or more,at least 250% or more, at least 300% or more, at least 350% or more, atleast 400% or more, at least 500% or more, at least 600% or more, atleast 700% or more, at least 800% or more, at least 900% or more, atleast 1000% or more at least 1500% or more or at least 2000% or more ofthe activity thereof.

In one aspect of the invention, the non-naturally occurring peptide Amay comprise any one or more of the peptide sequences set forth in SEQ.ID. NO. 89, 92, 98, 103 or 106, or a variant thereof being at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% similar thereto andhaving at least 10% or more, at least 15% or more, at least 20% or more,at least 25% or more, at least 30% or more, at least 35% or more, atleast 40% or more, at least 45% or more, at least 50% or more, at least55% or more, at least 60% or more, at least 65% or more, at least 70% ormore, at least 75% or more, at least 80% or more, at least 85% or more,at least 90% or more, at least 95% or more, at least 100%, at least 200%or more, at least 250% or more, at least 300% or more, at least 350% ormore, at least 400% or more, at least 500% or more, at least 600% ormore, at least 700% or more, at least 800% or more, at least 900% ormore, at least 1000% or more at least 1500% or more or at least 2000% ormore of the activity thereof.

Transfection Enhancing Agents

The complexes formed between the non-naturally occurring peptide, thenucleic acid and the transfection agent may be further enhanced byinclusion of moieties such as proteins or peptides that function fornuclear or other sub-cellular localization, function for transport ortrafficking, are receptor ligands, comprise cell-adhesive signals,cell-targeting signals, cell-internalization signals or endocytosissignals as well as peptides or functional portions thereof of viralfusogenic proteins of enveloped viruses, of viral nuclear localizationsignals, of receptor-ligands, of cell adhesion signals, ofcell-targeting signals or of internalization- or endocytosis-triggeringsignals.

The complex may also optionally contain a transfection enhancing agent,such as a nuclear localization protein or peptide, a fusogenic peptideor protein, receptor-ligand peptide or protein, a transport peptide orprotein, or a viral peptide or protein that is distinct in amino acidsequence from the non-naturally occurring peptides of the presentinvention. The suitable viral peptide may be derived from a virus suchas an influenza virus, a vesicular stomatitis virus, an adenovirus, analphavirus, a Semliki Forest Virus, a hepatitis virus, a herpes virus,an HIV virus, or a simian virus. The transfection enhancing agent mayalso be, for example, insulin, a transferrin, a epidermal growth factor,a fibroblast growth factor, a cell targeting antibody, a lactoferrin, afibronectin, an adenovirus penton base, Knob, a hexon protein, avesicular stomatitis virus glycoprotein, a Semliki Forest Virus coreprotein, a influenza hemagglutinin, a hepatitis B core protein, an HIVTat protein, a herpes simplex virus VP22 protein, a histone protein, anarginine rich cell permeability protein, a high mobility group protein,and invasin protein, and internalin protein, an endotoxin, a diptheriatoxin, a shigella toxin, a melittin, a magainin, a gramicidin, acecrophin, a defensin, a protegrin, a tachyplesin, a thionin, aindolicidin, a bactenecin, a drosomycin, an apidaecin, a cathelicidin, abacteriacidal-permability-increasing protein, a nisin, a buforin, orfragments thereof. The transfection enhancing agent may be chloroquine,a lysosomotrophic compound or any derivatives, variants, or combinationsthereof. The transfection agent may contain multimers of the same ordifferent peptides or proteins.

Any proteins or peptides (or fragments or portions thereof) of theinvention may be used in accordance with this invention, either singlyor in combination with other proteins or peptides. In a preferredaspect, two or more, three or more, four or more, five or more, six ormore, etc. proteins and/or peptides are used in the invention.Additionally, such single or multiple proteins and/or peptides may beused in combination with one or more, two or more, three or more, fouror more, five or more, six or more, etc. transfection agents. In anotherpreferred aspect, at least two peptides and/or proteins are used incombination with a transfection agent, preferably at least twotransfection agents such as lipids, and/or polycations such asdendrimers or PEI.

Further embodiments of the present invention are directed totransfection complexes containing the non-naturally occurring peptidesdescribed above in combination with one or more transfection reagents,which transfection reagents may include one or more cationic lipids, andoptionally one or more helper lipids. In some embodiments, atransfection complex may include a cargo to be delivered to the interiorof a cell, or optionally may be administered to an animal or to a humanpatient who would benefit from the administration thereof. Preferredthough non-limiting cargo molecules suitable for use with the presentinvention include nucleic acid molecules such as DNA molecules or RNAmolecules. Suitable DNA molecules may include a DNA molecule having anexpressible nucleic acid sequence, such as an expression vector or acDNA molecule comprising an open reading frame encoding a protein. Othersuitable molecules that may function as suitable cargo in the practiceof the present invention include RNA molecules, such as an mRNA moleculeor an RNAi molecule.

Methods of Making Peptides:

The non-naturally occurring peptides of the present invention can beproduced by any previously known peptide synthesis methods known tothose possessing ordinary skill level in the art, without limitation,including recombinant methods or peptide synthesis chemistry, such as,e.g., sold phase peptide synthesis. The solid phase synthesis method(Marrifield, J. Am. Chem. Soc., 85, 2149-2154, 1963) can be noted asmerely an example of such a peptide synthesis method. At present thepeptide can be produced simply and in a relatively short period of timeusing an automated, general purpose peptide synthesizer based on thoseprinciples. Additionally, the peptide can be produced using well-knownrecombinant protein production techniques, which techniques are widelyknown to the skilled artisan.

Transfection Reagents

The present invention also provides a transfection complex comprising,in non-covalent association, a non-naturally occurring peptide accordingto the present invention as described above and incorporated herein, atleast one cargo molecule as defined above and incorporated herein, atleast one transfection reagent as defined above and incorporated herein.

In certain preferred though non-limiting embodiments, a transfectionreagent selected for use in the practice of the present invention mayinclude one or more cationic lipids. In some embodiments, the one ormore cationic lipids may optionally include at least one, optionallymore than one neutral lipid or helper lipid.

In some embodiments, a transfection reagent may include one or morelipids of which one or more can be cationic lipids. In some embodiments,the transfection reagent may include a mixture of neutral and cationiclipids. In some embodiments, the transfection reagent may include one ormore peptides and/or proteins which are distinct from the non-naturallyoccurring peptide of the present invention and which can be providedalone or in admixture with one or more lipids. By way of non-limitingexample, one such peptide may include a reagent such as, e.g., PLUS™Reagent (Life Technologies, Carlsbad, Calif.). In some preferredembodiments, the transfection reagent forms a non-covalent complex withthe macromolecule/cargo to be delivered to the interior of the cell, thenon-naturally occurring peptides of the present invention, andoptionally the one or more helper or neutral lipids. In preferredembodiments, transfection complexes made in accordance with the methodsdescribed herein may have a net positive charge, thereby facilitatingthe interaction of the transfection complex with the cell membrane.

In some embodiments, a transfection reagent suitable for use inaccordance with the present invention may be any material, formulationor composition known to those of skill in the art that facilitates theentry of a macromolecule into a cell. In some embodiments, thetransfection reagent can be any compound and/or composition thatincreases the uptake of one or more nucleic acids or other cargomolecules into one or more target cells.

A variety of transfection reagents are known to those skilled in theart. Suitable transfection reagents can include, but are not limited to,one or more compounds and/or compositions comprising cationic polymerssuch as polyethyleneimine (PEI), polymers of positively charged aminoacids such as polylysine and polyarginine, positively charged dendrimersand fractured dendrimers, cationic β-cyclodextrin containing polymers(CD-polymers), DEAE-dextran and the like. In some embodiments, a reagentfor the introduction of macromolecules into cells can comprise one ormore lipids which can be cationic lipids and/or neutral lipids.Preferred lipids include, but are not limited to,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamonium chloride (DOTMA),dioleoylphosphatidylcholine (DOPE),1,2-Bis(oleoyloxy)-3-(4′-trimethylammonio) propane (DOTAP),1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol (DOTB),1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl(4′-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide(CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),0,0′-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammoniumchloride, spermine conjugated to one or more lipids (for example,5-carboxyspermylglycine dioctadecylamide (DOGS),N,N^(I),N^(II),N^(III)-tetramethyl-N,N^(I),N^(II),N^(III)-tet-rapalmitylspermine(TM-TPS) and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde(DPPES)), lipopolylysine (polylysine conjugated to DOPE), TRIS(Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fatty acids(TFAs) and/or peptides such as trilysyl-alanyl-TRIS mono-, di-, andtri-palmitate, (3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol(DC-Chol), N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride(TMAG), dimethyl dioctadecylammonium bromide (DDAB),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-iniumtrifluoroacetate(DOSPA) and combinations thereof.

Those skilled in the art will appreciate that certain combinations ofthe above mentioned lipids have been shown to be particularly suited forthe introduction of nucleic acids into cells for example a 3:1 (w/w)combination of DOSPA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTAMINE™, a 1:1(w/w) combination of DOTMA and DOPE is available from Life TechnologiesCorporation, Carlsbad, Calif. under the trade name LIPOFECTIN®, a 1:1(M/M) combination of DMRIE and cholesterol is available from LifeTechnologies Corporation, Carlsbad, Calif. under the trade name DMRIE-Creagent, a 1:1.5 (M/M) combination of TM-TPS and DOPE is available fromLife Technologies Corporation, Carlsbad, Calif. under the trade nameCELLFECTIN® and a 1:2.5 (w/w) combination of DDAB and DOPE is availablefrom Life Technologies Corporation, Carlsbad, Calif. under the tradename LIPOFECTACE®. In addition to the above-mentioned lipidcombinations, other formulations comprising lipids in admixture withother compounds, in particular, in admixture with peptides and proteinscomprising nuclear localization sequences, are known to those skilled inthe art. For example, see international application no. PCT/US99/26825,published as WO 00/27795, both of which are incorporated by referenceherein.

Lipid aggregates such as liposomes have been found to be useful asagents for the delivery of macromolecules into cells. In particular,lipid aggregates comprising one or more cationic lipids have beendemonstrated to be extremely efficient at the delivery of anionicmacromolecules (for example, nucleic acids) into cells. One commonlyused cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).Liposomes comprising DOTMA alone or as a 1:1 mixture withdioleoylphosphatidylethanolamine (DOPE) have been used to introducenucleic acids into cells. A 1:1 mixture of DOTMA:DOPE is commerciallyavailable from Life Technologies Corporation, Carlsbad, Calif. under thetrade name of LIPOFECTIN™. Another cationic lipid that has been used tointroduce nucleic acids into cells is1,2-bis(oleoyl-oxy)-3-3-(trimethylammonia) propane (DOTAP). DOTAPdiffers from DOTMA in that the oleoyl moieties are linked to thepropylamine backbone via ether bonds in DOTAP whereas they are linkedvia ester bonds in DOTMA. DOTAP is believed to be more readily degradedby the target cells. A structurally related group of compounds whereinone of the methyl groups of the trimethylammonium moiety is replacedwith a hydroxyethyl group are similar in structure to the Rosenthalinhibitor (RI) of phospholipase A (see Rosenthal, et al., (1960) J.Biol. Chem. 233:2202-2206.). The RI has stearoyl esters linked to thepropylamine core. The dioleoyl analogs of RI are commonly abbreviatedDOR1-ether and DOR1-ester, depending upon the linkage of the lipidmoiety to the propylamine core. The hydroxyl group of the hydroxyethylmoiety can be further derivatized, for example, by esterification tocarboxyspermine.

Another class of compounds which has been used for the introduction ofmacromolecules into cells comprise a carboxyspermine moiety attached toa lipid (see, Behr, et al., (1989) Proceedings of the National Academyof Sciences, USA 86:6982-6986 and EPO 0 394 111). Examples of compoundsof this type include dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES) and 5-carboxyspermylglycinedioctadecylamide (DOGS). DOGS is commercially available from PROMEGA™,Madison, Wis. under the trade name of TRANSFECTAM™.

A cationic derivative of cholesterol(3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol, DC-Chol) hasbeen synthesized and formulated into liposomes with DOPE (see Gao, etal., (1991) BBRC 179(1):280-285.) and used to introduce DNA into cells.The liposomes thus formulated were reported to efficiently introduce DNAinto the cells with a low level of cellular toxicity. Lipopolylysine,formed by conjugating polylysine to DOPE (see Zhou, et al., (1991) BBA1065:8-14), has been reported to be effective at introducing nucleicacids into cells in the presence of serum.

Other types of cationic lipids that have been used to introduce nucleicacids into cells include highly packed polycationic ammonium, sulfoniumand phosphonium lipids such as those described in U.S. Pat. Nos.5,674,908 and 5,834,439, and international application no.PCT/US99/26825, published as WO 00/27795.

One non-limiting transfection reagent for delivery of macromolecules inaccordance with the present invention is LIPOFECTAMINE 2000™ orderivatives thereof which is available from Life technologies (see U.S.international application no. PCT/US99/26825, published as WO 00/27795).

Another preferred though non-limiting transfection reagent suitable fordelivery of macromolecules to a cell is EXPIFECTAMINE™ or derivativesthereof.

Other suitable transfection reagents include LIPOFECTAMINE® RNAiMAX,LIPOFECTAMINE® LTX, OLIGOFECTAMINE® CELLFECTIN™, INVIVOFECTAMINE®,INVIVOFECTAMINE® 2.0, and any of the lipid reagents or formulationsdisclosed in U.S. Patent Appl. Pub. No. 2012/0136073, by Yang et al.(incorporated herein by reference thereto). A variety of othertransfection reagents are known to the skilled artisan and may beevaluated for the suitability thereof to the transient transfectionsystems and methods described herein.

Various preferred though non-limiting cationic lipids and transfectionreagent suitable for use with the present invention will now bedescribed in greater detail below. It should be noted however, that theexplicit disclosure of one or more specific cationic lipids, or one ormore genera of cationic lipids, is not meant to preclude the use ofother reagents or lipids that are capable of being used in conjunctionwith the non-naturally occurring peptides of the present invention, andthat the selection of alternative cationic lipids or transfectionsreagents, and the use thereof in the context of the present invention,is well within the purview of the skilled art, and that such a personmay readily use such a reagent without departing from the spirit andscope of the present invention.

Some embodiments of the present invention provide lipid aggregatescomprising one or more non-naturally occurring peptides described abovein combination with one or more cationic lipids. Without being limitedto or bound by any theory or mechanistic explanation for the performanceof the composition forming the basis of the present invention, andsolely in the interest of providing complete disclosure thereof, it isbelieved that the non-naturally occurring peptides of the presentinvention, when used in combination with one or more transfectionreagents, in particular with one or more cationic transfection lipids,improve ability of a transfection complex comprising a lipid aggregateand the cargo molecule to be delivered to the interior of a cell. Theuse of cationic lipids, optionally in conjunction with one or morehelper lipids or one or more neutral lipids, may allow for greaterencapsulation of the cargo molecule by the lipid aggregate and furthermay assist with the fusion of the liposomal lipid aggregate with thetarget cell membrane, thereby improving enhancing the delivery of thecargo molecule.

Cationic lipids useful for use in the formation of transfectioncomplexes of the present invention can be either monovalent orpolyvalent cationic lipids or a mixture of cationic lipids. Ofparticular interest are cationic lipids recognized in the art as usefulin transfection methods, including, but not limited to, DOTMA, DOTAP,DDAB, DMRIE, DOSPA, DOSPER, TMTPS, DHMS, DHDMS and their analogs orhomologs. Optionally, the lipid aggregate may further comprise at leastone additional helper lipid. Helper lipids are known in the art andinclude, but are not limited to, neutral lipids, preferably selectedfrom the group consisting of DOPE, DOPC and cholesterol. Optionally,transfection complexes of the present invention may include commerciallyavailable transfection reagents containing cationic lipids such asLipofectin®, LIPOFECTAMINE™ RNAiMAX and LIPOFECTAMINE™ 2000,LIPOFECTAMINE® 3000, LIPOFECTAMINE® LTX (Life Technologies Corporation,Carlsbad, Calif.).

A further embodiment of the invention provides a cationic lipidaggregate, comprising one or more cationic lipids, optionally one ormore helper lipids, and one or more cargo molecules complexed with oneor more of the non-naturally occurring peptides described above. Thecargo molecule may be any substance that is to be conveyed to theinterior of a cell, either in culture in a laboratory or in a tissue inan animal or a human. The cargo may, depending on the application, be amacromolecule such as a nucleic acid, a protein, or a peptide, or may bea drug or other organic small molecule. In some embodiments, thepreferred cargo for forming a transfection complex is a nucleic acidsuch as, e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).In some embodiments, the preferred cargo may be a DNA molecule. The DNAcan be either linear DNA or circular DNA, such as DNA in the form of acircular plasmid, an episome or an expression vector. In certainpreferred though non-limiting embodiments, the term macromolecule refersto complementary DNA (cDNA) have an expressible nucleic acid sequence,including at least one open reading frame operably linked to one or morenucleic acid sequence required for the transcription of an mRNA from theexpressible nucleic acid sequence. In other embodiments, a preferredcargo may be an RNA molecule. The RNA molecule may be any type of RNAmolecule, without limitation, including but not limited to an mRNA, ansiRNA, an miRNA, an antisense RNA, a ribozyme, or any other type orspecies of RNA molecule familiar to those skilled in the art withoutlimitation, that would be sought to be delivered to the interior of acell.

Preferably the transfection complex of the present invention may includea non-naturally occurring peptide as described above, at least onecargo, at least on cationic lipid, and optionally at least one helperlipid. The transfection complex, one formed, is stable in aqueoussolution and can either be contacted with a cell or a tissue in a humanor an animal immediately after being formed, or can be stored for aperiod prior to being contacted with the cell or tissue. Thetransfection complex is stable and can be stored for a time period of atleast 30 minutes, at least 45 minutes, at least 1 hour, at least 2hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10hours, at least 15 hours, at least 20 hours, at least 24 hours, at least48 hours, at least 72 hours, at least 5 days, at least 7 days, at least14 days, at least 28 days, at least 1 month, at least 2 months, at least3 months, at least 4 months, at least 5 months, at least 6 months or atleast 1 year. It is understood, that the storage period can be betweenany of these time periods, for example between 31 minutes and 1 hour orbetween 1 hour and 24 hours.

Generally, the transfection complexes of this invention can comprise anycationic lipid, either monovalent or polyvalent, including those inknown transfection reagents (see Table 5). Cationic lipids includesaturated and unsaturated alkyl and alicyclic ethers and esters ofamines, amides or derivatives thereof. Straight-chain and branched alkyland alkene groups of cationic lipids can contain from 1 to about 25carbon atoms. Preferred straight-chain or branched alkyl or alkenegroups have six or more carbon atoms. More preferred straight-chain orbranched alkyl or alkene groups have eight to about twenty carbon atoms.Alicyclic groups can contain from about 6 to 30 carbon atoms, and morepreferably eight to twenty carbon atoms. Preferred alicyclic groupsinclude cholesterol and other steroid groups. Cationic lipids can beprepared with a variety of counter ions (anions) including among others:Cl—, Br—, I—, F—, acetate, trifluoroacetate, sulfate, nitrite, triflate,and nitrate.

In the lipid aggregates of this invention, cationic lipids canoptionally be combined with non-cationic lipids, preferably neutrallipids, to form lipid aggregates that bind to themodified-peptide-nucleic acid complex. Neutral lipids useful in thisinvention as helper lipids include, among many others: lecithins (andderivatives thereof); phosphotidylethanolamine (and derivativesthereof); phosphatidylethanolamines, such as DOPE(dioleoylphosphatidylethanolamine), DphPE(diphytanoylphosphatidyl-ethanolamine), DPPE(dipalmitoylphosphatidylethanolamine),dipalmiteoylphosphatidyl-ethanolamine, POPE(palmitoyloleoylphosphatidylethanolamine) anddistearoyl-phosphatidylethanolamine; phosphotidylcholine;phosphatidylcholines, such as DOPC (dioleoylphosphidylcholine), DPPC(dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;phosphatidyl-glycerol; phosphatidylglycerols, such as DOPG(dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol),and distearoylphosphatidylglycerol; phosphatidyl-serine (and derivativesthereof); phosphatidylserines, such as dioleoyl- ordipalmitoylphosphatidylserine; diphosphatidylglycerols; fatty acidesters; glycerol esters; sphingolipids; cardolipin; cerebrosides; andceramides; and mixtures thereof. Neutral lipids also include cholesteroland other 3ßOH-sterols as well as derivatives thereof.

The following patent documents, patent applications, or references areincorporated by reference herein in their entirety and in particular fortheir disclosure of transfection agents containing cationic and neutral(helper) lipids which may be used to comprise the lipid aggregates ofthe present invention in conjunction with the cationic lipids: U.S. Pat.Nos. 6,075,012; 6,020,202; 5,578,475; 5,736,392; 6,051,429; 6,376,248;5,334,761; 5,316,948; 5,674,908; 5,834,439; 6,110,916; 6,399,663;6,716,882; 5,627,159; PCT/US/2004/000430, published as WO 04063342 A2;PCT/US/9926825, published as WO 0027795 A1; PCT/US/04016406, publishedas WO 04105697; and PCT/US2006/019356, published as WO 07130073 A2.Table 5 also lists transfection agents comprising cationic lipids andneutral lipids which may be used to comprise the lipid aggregates of thepresent invention in conjunction with the cationic lipids.

TABLE 5 Non-limiting Examples of Transfection Reagents TransfectionPatents and/or Agent Description Citations available from BMOPN-(2-bromoethyl)-N,N- dimethyl-2,3-bis(9- octadecenyloxy)-propana miniumbromide) BMOP:DOPE 1:1 (wt/wt) formulation Walzem et al., Poult ofN-(2-bromoethyl)- Sci. 76: 882-886, N,N-dimethyl-2,3-bis(9- 1997.Transfection of octadecenyloxy)-propana avian LMH-2A minium bromide)hepatoma cells with (BMOP) and DOPE cationic lipids. Cationic Cationicpolysaccharides Published U.S. patent polysaccharides application Ser.No. 2002/0146826 CellFECTIN ® 1:1.5 (M/M) formulation U.S. Pat. Nos.Invitrogen of N,NI,NII,NIII- 5,674,908, 5,834,439 tetramethyl-N,NI,NII,and 6,110,916 NIII-tetrapalmitylspermine (TM-TPS) and dioleoylphosphatidylethanolamine (DOPE) CTAB:DOPE formulation of cetyltrimethyl-ammonium bromide (CATB) and dioleoylphosphatidylethanol- amine (DOPE)Cytofectin GSV 2:1 (M/M) formulation of (*Cytofectin GS cytofectin GS*and corresponds to dioleoyl phosphatidyl- Gilead ethanolamine (DOPE)Sciences' GS 3815) DC-Cholesterol 3,β-N,(N′,N′- (DC-Chol)dimethylaminoethane)- carbamo-yl]cholesterol DC-Chol:DOPE formulation of3,β- Gao et al., Biochim. N,(N′,N′- Biophys. Res. Comm.dimethylaminoethane)- 179: 280-285, 1991 carbamo-yl]cholesterol(DC-Chol) and dioleoyl phosphatidyl- ethanolamine (DOPE) DC-6-14O,O′-Ditetradecanoyl-N- Kikuchi et al., Hum (alpha- Gene Ther 10: 947-trimethylammonioacetyl) 955, 1999. diethan olamine chloride Developmentof novel cationic liposomes for efficient gene transfer into peritonealdisseminated tumor. DCPE Dicaproylphosphtidylethanol- amine DDPESDipalmitoylphosphatidyl- Behr et al., Proc. ethanolamine 5- Natl. Acad.Sci. USA carboxyspermylamide 86: 6982-6986, 1989. Efficient genetransfer into mammalian primary endocrine cells withlipopolyamine-coated DNA; EPO published patent application 0 394 111DDAB didoceyl methylammonium bromide Dextran and DEAE-Dextran; DextranMai et al., J Biol dextran derivatives sulfate Chem. 277: 30208- orconjugates 30218, 2002. Efficiency of protein transduction is celltype-dependent and is enhanced by dextran sulfate. Diquaternary(examples 

 N′N′- Rosenzweig et al., Vical ammonium salts dioleyl-N,N′N″N′-Bioconjug Chem tetramethyl-1,2- 12: 258-263, 2001. ethanediamineDiquaternary (TmedEce), N′N′- ammonium dioleyl-N,N′N″N′- compounds astetramethyl-1,3- transfection agents; propanediamine U.S. Pat. No.5,994,317 (PropEce), N′N′-dioleyl- N,N′N″N′-tetramethyl-1,6-hexanediamine (HexEce), and their corresponding N′N′- dicetylsaturated analogues (TmedAce, PropAce and HexAce) DLRIE dilauryloxypropyl-3- Felgner et al., Ann Vical dimethylhydroxy NY Acad Sci 772:126- ethylammonium bromide 139, 1995. Improved cationic lipidformulations for in vivo gene therapy. DMAP 4-dimethylaminopyridine DMPEDimyristoylphospatidylethanol- amine DMRIE N-[1-(2,3 Konopka et al.,dimyristyloxy)propyl]- Biochim Biophys N,N-dimethyl-N-(2- Acta 1312:186-96, hydroxyethyl) 1996. ammonium bromide Huma55mmuneno- deficiencyvirus type- 1 (HIV-1) infection increases the sensitivity of macrophagesand THP-1 cells to cytotoxicity by cationic liposomes. DMRIE-C 1:1formulation of N-[1- U.S. Pat. Nos. Invitrogen (2,3- 5,459,127 anddimyristyloxy)propyl]- 5,264,618, to Feigner, N,N-dimethyl-N-(2- et al.(Vical) hydroxyethyl) ammonium bromide (DMRIE) and cholesterolDMRIE:DOPE formulation of 1,2- San et al., Hum Genedimyristyloxypropyl-3- Ther 4: 781-788, dimethyl-hydroxyethyl 1993.Safety and ammonium bromide and short-term toxicity of dioleoylphosphatidyl- a novel cationic lipid ethanolamine (DOPE) formulation forhuman gene therapy. DOEPC Dioleoylethyl- phosphocholine DOHME N-[1-(2,3-dioleoyloxy)propyl]-N- [1-(2-hydroxyethyl)]- N,N-dimethylammonium iodideDOPC Dioleoylphosphatidylcholine DOPC:DOPS 1:1 (wt %) formulation ofAvanti DOPC (dioleoylphosphatidylcholine) and DOPS DOSPA2,3-dioleoyloxy-N-[2- (sperminecarboxamidoethyl]- N,N-di-met-hyl-1-propanaminium trifluoroacetate DOSPA:DOPE Formulation of 2,3- Baccagliniet al., J dioleoyloxy-N-[2- Gene Med 3: 82-90,(sperminecarboxamidoethyl]- 2001. Cationic N,N-di-met-hyl-1-liposome-mediated propanaminium gene transfer to rat trifluoroacetate(DOSPA) salivary epithelial and dioleoyl cells in vitro and inphosphatidyl- vivo. ethanolamine (DOPE) DOSPER 1,3-Di-Oleoyloxy-2-(6-Buchberger et al., Roche Carboxy-spermyl)- Biochemica 2: 7-10,propylamid 1996. DOSPER liposomal transfection reagent: a reagent withunique transfection properties. DOTAP N-[1-(2,3- dioleoyloxy)propyl] -N,N,N-trimethyl- ammonium methylsulfate DOTMA N-[1-(2,3-dioleyloxy)propyl]-n,n,n- trimethylammoniumchloride DPEPCDipalmitoylethylphosphatidyl- choline Effectene (non-liposomal lipidZellmer et al., Qiagen formulation used in Histochem Cell Biolconjunction with a 115: 41-47, 2001. special DNA-condensing Long-termexpression enhancer and optimized of foreign genes in buffer) normalhuman epidermal keratinocytes after transfection with lipid/DNAcomplexes. FuGENE ® 6 Wiesenhofer et al., J Roche Neurosci Methods 92:145-152, 1999. Improved lipid- mediated gene transfer in C6 glioma cellsand primary glial cells using FuGene. GAP- N-(3-aminopropyl)-N,N-Stephan et al., Hum DLRIE:DOPE dimethyl-2,3- Gene Ther 7: 1803-bis(dodecyloxy)-1- 1812, 1996. A new propaniminium cationic liposomebromide/dioleyl DNA complex phosphatidylethanolamine enhances theefficiency of arterial gene transfer in vivo. GS 2888 cytofectin Lewiset al., Proc Gilead Sciences Natl Acad Sci USA 93: 3176-3181, 1996. Aserum-resistant cytofectin for cellular delivery of antisenseoligodeoxynucleotides and plasmid DNA. Lipofectin ® 1:1 (w/w)formulation of U.S. Pat. Nos. Invitrogen N-(1-2,3- 4,897,355; 5,208,066;dioleyloxypropyl)- and 5,550,289. N,N,N- triethylammonium (DOTMA) anddioleylphosphatidyl- ethanolamine (DOPE) LipofectACE ™ 1:2.5 (w/w)formulation Invitrogen of dimethyl dioctadecylammonium bromide (DDAB)and dioleoyl phosphatidylethanolamine (DOPE) LIPOFECTAMINE ® U.S. Pat.No. Invitrogen LTX 7,915,230 LIPOFECTAMINE ™ 3:1 (w/w) formulation ofU.S. Pat. No. Invitrogen 2,3-dioleyloxy-N- 5,334,761; and U.S.[2(sperminecarboxamido)ethyl]- Pat. Nos. 5,459,127 and N,N-dimethyl-1-5,264,618, to Felgner, propanaminium et al. (Vical) trifluoroacetate(DOSPA) and dioleoyl phosphatidylethanolamine (DOPE) LIPOFECTAMINE ™2000 Invitrogen LipofectAMlNE U.S. Pat. Nos. Invitrogen PLUS ™ 5,736,392and 6,051,429 LIPOFECTAMINE ® 3000 Invitrogen LipoTAXI ® Stratagenemonocationic (examples:) 1-deoxy-1- Banerjee et al., J Med transfectionlipids [dihexadecyl(methyl)ammonio]- Chem 44: 4176-4185, D-xylitol;1-deoxy-1- 2001. Design, [methyl(ditetradecyl)ammonio]- synthesis, andD-arabinitol; 1-deoxy-1- transfection biology[dihexadecyl(methyl)ammonio]- of novel cationic D-arabinitol; 1-deoxy-1-glycolipids for use in [methyl(dioctadecyl)ammonio]- liposomal geneD-arabinitol delivery. O-Chol 3 beta[l-ornithinamide- Lee et al., GeneTher carbamoyl] cholesterol 9: 859-866, 2002. Intraperitoneal genedelivery mediated by a novel cationic liposome in a peritonealdisseminated ovarian cancer model. OliogfectAMINE ™ InvitrogenPiperazine based Piperazine based U.S. Pat. Nos. Vical amphilic cationicamphilic cationic lipids 5,861,397 and lipids 6,022,874 PolyFect(activated-dendrimer Qiagen molecules with a defined sphericalarchitecture) Protamine Protamine mixture Sorgi et al., Gene Sigmaprepared from, e.g., Ther 4: 961-968, salmon, salt herring, etc.; 1997.Protamine can be supplied as, e.g., a sulfate enhances sulfate orphosphate. lipid-mediated gene transfer. SuperFect (activated-dendrimerTang et al., Qiagen molecules with a defined Bioconjugate Chem.spherical architecture) 7: 703, 1996. In vitro gene delivery by degradedpolyamido- amine dendrimers.; published PCT applications WO 93/19768 andWO 95/02397 Tfx ™ N,N,N′,N′-tetramethyl- Promega N,N′-bis(2-hydroxyethyl)-2,3- di(oleoyloxy)-1,4- butanediammonium iodide] and DOPETransFast ™ N,N [bis (2- Promega hydroxyethyl)-N-methyl- N-[2,3-di(tetradecanoyloxy) propyl] ammonium iodide and DOPE TransfectAceInvitrogen TRANSFECTAM ™ 5- Behr et al., Proc. Promegacarboxylspermylglycine Natl. Acad. Sci. USA dioctadecylamide 86:6982-6986, 1989; (DOGS) EPO Publication 0 394 111 TransMessenger(lipid-based formulation Qiagen that is used in conjunction with aspecific RNA- condensing enhancer and an optimized buffer; particularlyuseful for mRNA transfection) Vectamidine 3-tetradecylamino-N-tert-Ouahabi et al., FEBS butyl-N′- Lett 414: 181-92,tetradecylpropionamidine 1997. The role of (a.k.a. diC14-amidine)endosome destabilizing activity in the gene transfer process mediated bycationic lipids. X-tremeGENE ™ Roche

In some preferred though non-limiting embodiments, lipid aggregates caninclude at least a first cationic lipid and optionally at least a firstneutral lipid, wherein said lipid aggregate is suitable for forming acationic complex with a nucleic acid under aqueous conditions, whereinsaid the cationic lipids have the structure:

and salts thereof; where:

R₁ and R₂, independently, are an alkyl, alkenyl or alkynyl groups,having from 8 to 30 carbon atoms;

an alkyl, alkenyl or alkynyl groups, having from 8 to 30 carbon atomsand optionally substituted by one or more of an alcohol, anaminoalcohol, an amine, an amide, an ether, a polyether, an ester, amercaptan, alkylthio, or a carbamoyl group or where R₁ is—(CH₂)_(q)—N(R₆)_(t)R₇R₈;

R₃ and R₄, independently, are hydrogens, or alkyl, alkenyl or alkynylgroups having from 8 to 30 carbon atoms and optionally substituted byone or more of an alcohol, an aminoalcohol, an amine, an amide, anether, a polyether, an ester, a mercaptan, alkylthio, or a carbamoylgroup;

R₅-R₈, independently, are hydrogens, or alkyl, alkenyl or alkynylgroups;

R₉ is a hydrogen, or an alkyl, alkenyl or alkynyl group, a carbohydrateor a peptide;

r, s and t are 1 or 0 to indicate the presence or absence of theindicated R group, when any of r, s or t are 1 the nitrogen to which theindicated R group is attached is positively charged and wherein at leastone of r, s or t is 1;

q is an integer ranging from 1 to 6, inclusive;

X^(v−) is an anion, where v is the valency of the anion and A is thenumber of anions;

L is a divalent organic radical capable of covalently linking the twonitrogens selected from:(CH₂)_(n), where n is an integer ranging from 1to 10, inclusive, which is optionally substituted with one or more ZR₁₀groups, where Z is O or S, and R₁₀ is hydrogen or an alkyl, alkenyl oralkynyl group; or

{—(CH₂)_(k)—Y—(CH₂)_(m)}_(p)—, where k and m, independently, areintegers ranging from 1 to 10, inclusive, and p is an integer rangingfrom 1 to 6, inclusive, and Y is O, S, CO, COO, CONR₁₁, NR₁₁CO, orNR₁₁COR₁₁N where R₁₁, independent of any other R₁₁, is hydrogen or analkyl group;

wherein one or more CH₂ groups of the alkyl, alkenyl or alkynyl groupsof R₁-R₁₀ can be replaced with an O, S, S—S, CO, COO, NR₁₂CO, NR₁₂COO,or NR₁₂CONR₁₂ where R₁₂, independent of any other R₁₂, is hydrogen or analkyl, alkenyl or alkynyl group; and

wherein the alkyl, alkenyl or alkynyl groups of R₁-R₁₂ are optionallysubstituted with one or more OR₁₃, CN, halogens, N(R₁₃)₂, peptide, orcarbohydrate groups where R₁₃, independently of other R₁₃, is hydrogenor an alkyl, alkenyl or alkynyl group, and

wherein at least one of R₃ and R₄, when present as alkyl groups, aresubstituted with both OR₁₃ and N(R₁₃)₂ groups.

The synthesis of these compounds and methods for the preparation oflipid aggregates incorporating same may be achieved by any means knownto those skilled in the art without limitation. Exemplary thoughnon-limiting methods to synthesize such compounds, and methods for theformation of lipid aggregates incorporating same, may be found in, forexample, U.S. Pat. No. 7,166,745 and PCT Publication No. WO 00/27795,both of which are expressly incorporated by reference in their entiretyas though fully set forth herein.

In some embodiments, the lipid aggregates that form the basis of thepresent invention may further optionally include one, optionally morethan one additional cationic lipid selected from the list consisting ofTMTPS, DOGS, DPPES, DOTMA, DOTAP, DDAB, DMRIE, DOSPA, and DOSPER.

In some embodiments of the present lipid aggregates, a particularlypreferred though non-limiting cationic lipid used in the formation ofthe inventive transfection complexes may bedihydroxyl-dimyristylspermine tetrahydrochloride (hereinafter referredto as “DHDMS”) having the structure:

In some embodiments of the present lipid aggregates, a particularlypreferred though non-limiting cationic lipid used in the formation ofthe inventive transfection complexes may be hydroxyl-dimyristylsperminetetrahydrochloride (hereinafter referred to as “HDMS”) having thestructure:

In some embodiments, the neutral lipids may be selected from thefollowing; DOPE, cholesterol or DOPC. In one embodiment, a neutral lipidmay be one of cholesterol, DOPE or DOPC. In an embodiment, the a lipidis cholesterol. In an embodiment, a neutral lipid is DOPE. In anembodiment, a lipid is DOPC.

In one embodiment, the optional second neutral lipid maybe one ofcholesterol, DOPE or DOPC, except that the second neutral lipid and thefirst neutral lipid escribed above are not the same. In an embodiment,the optional second neutral lipid is cholesterol. In an embodiment, theoptional second neutral lipid is DOPE. In an embodiment, the optionalsecond neutral lipid is DOPC.

In some embodiments, the molar ratio of the cationic lipid in the lipidaggregate may be in the range of about 0.1 to about 0.8. In someembodiments, the molar ratio of the cationic lipid in the lipidaggregate may be between 0.1 to about 0.2, about 0.15 to about 0.25,about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 to about0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45 toabout 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about 0.6to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, or about0.75 to about 0.85.

In some embodiments, the molar ratio of DHDMS in the lipid aggregate maybe in the range of about 0.1 to about 0.7. In some embodiments, themolar ratio of the cationic lipid in the lipid aggregate may be about0.1, about 0.2, about 0.25, about 0.3 or about 0.4, or any range fallingtherebetween.

In some embodiments, the molar ratio of DHDMS is about 0.1 to about 0.4.In some embodiments, the molar ratio of DHDMS is about 0.1, about 0.2,about 0.25, about 0.3, about 0.4, or any range falling therebetween.

In some embodiments, the molar ratio of HDMS in the lipid aggregate maybe in the range of about 0.1 to about 0.4. In some embodiments, themolar ratio of the second cationic lipid in the lipid aggregate may beabout 0.1, about 0.2, about 0.25, about 0.3 or about 0.4, or any rangefalling therebetween.

In some embodiments, the molar ratio of HDMS is about 0.1 to about 0.4.In some embodiments, the molar ratio of HDMS is about 0.1, about 0.2,about 0.25, about 0.3, about 0.4, or any range falling therebetween.

In some embodiments, the molar ratio of the neutral lipid in the lipidaggregate may be in the range of about 0.1 to about 0.8. In someembodiments, the molar ratio of the cationic lipid in the lipidaggregate may be between 0.1 to about 0.2, about 0.15 to about 0.25,about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 to about0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45 toabout 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about 0.6to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, or about0.75 to about 0.85, or any range falling therebetween.

In some embodiments, the molar ratio of cholesterol is about 0.1 toabout 0.8. In some embodiments, the molar ratio of the cationic lipid inthe lipid aggregate may be between 0.1 to about 0.2, about 0.15 to about0.25, about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 toabout 0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45to about 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about0.6 to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, orabout 0.75 to about 0.85, or any range falling therebetween.

In some embodiments, the molar ratio of DOPE is about 0.1 to about 0.8.In some embodiments, the molar ratio of the cationic lipid in the lipidaggregate may be between 0.1 to about 0.2, about 0.15 to about 0.25,about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 to about0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45 toabout 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about 0.6to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, or about0.75 to about 0.85, or any range falling therebetween.

In some embodiments, the molar ratio of DOPC is about 0.1 to about 0.4.In some embodiments, the molar ratio of DOPC is about 0.1, about 0.2,about 0.25, about 0.3, about 0.4, or any range falling therebetween.

In some embodiments, the molar ratio of cholesterol is about 0.2 toabout 0.8. In some embodiments, the molar ratio of cholesterol is about0.1, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about0.75, about 0.8, or any range falling therebetween.

In some embodiments, the molar ratio of DOPE is about 0.2 to about 0.8.In some embodiments, the molar ratio of DOPE is about 0.1, about 0.2,about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5,about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, orany range falling therebetween.

In some embodiments, the molar ratio of DOPC is about 0.2 to about 0.8.In some embodiments, the molar ratio of DOPC is about 0.1, about 0.2,about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5,about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, orany range falling therebetween.

In some embodiments, the molar ratio of DHDMS is about 0.1, 0.2, 0.25,0.3, 0.4, or 0.5 and molar ratio of the neutral lipid is about 0.1, 0.2,0.25, 0.3, 0.4, or 0.5.

In some embodiments, the molar ratio of HDMS is about 0.1, 0.2, 0.25,0.3, 0.4, or 0.5 and molar ratio of neutral lipid is about 0.1, 0.2,0.25, 0.3, 0.4, or 0.5.

The composition of a variety of lipid formulations in accordance withseveral non-limiting embodiments of the invention are provided in TableI. The provision of these exemplary embodiments is in no way meant tolimit the scope of the invention solely to those formulations disclosed.On the contrary, it is merely meant to provide a variety of possiblelipid aggregate formulations that can be used in the practice of thepresent invention. Nevertheless, it will be apparent to one skilled inthe art that the formulations may be changed or altered, and additionalcomponents (such as, e.g., additional cationic or neutral lipids,peptide targeting moieties, and the like) may be added, or one of therecited neutral lipids set forth in Table I may optionally be removed,and the resulting formulations will be within the spirit and scope ofthe invention as described herein.

Preparation and Use of Complexes Containing Non-Naturally OccurringPeptides

Another embodiment of the present invention provides a method fordelivering a polyanion such as a nucleic acid molecule into a cell orcells, wherein the method comprises forming a lipid aggregate,preferably a liposome, comprising one or more cationic lipids and one ormore neutral lipids, contacting the lipid aggregate with the polyanionthat has already been complexed with the non-naturally occurring peptideof the present invention by virtue of the presence of the cationicregion B therein, thereby forming a neutral or positively chargedpolyanion-peptide-lipid aggregate complex, and incubating a cell orcells with the complex. Useful anions include proteins, peptides andnucleic acids, preferably DNA or RNA. Preferably, the lipid aggregatefurther comprises at least one additional helper lipid. Optionally, thepolyanion-lipid aggregate complex is stored for a period prior to beingcontacted with the cell or cells. The polyanion-lipid aggregate complexis stable and can be stored for a time period of at least 45 minutes, atleast 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 10 hours, at least 15 hours, at least 20 hours,at least 24 hours, at least 48 hours, at least 72 hours, at least 5days, at least 7 days, at least 14 days, at least 28 days, at least 1month, at least 2 months, at least 3 months, at least 4 months, at least5 months, at least 6 months or at least 1 year, or for a time periodbetween any of these time periods. This invention is particularly usefulto deliver RNAi, including siRNA, short hairpin RNA (shRNA), and smalltemporally regulated RNA (stRNA), which optionally are chemicallymodified.

The methods of the present invention involve contacting any cell,preferably a eukaryotic cell, with a transfection complex comprising atleast a non-naturally occurring peptide, a transfection agent and anucleic acid as described above. The complex optionally may also containone or more additional peptides or proteins, such as a fusogenic,membrane-permeabilizing, transport or traffickingsub-cellular-localization, or receptor-ligand peptide or protein. Theseadditional peptides or proteins optionally may be conjugated to anucleic acid-binding group, or optionally conjugated to the transfectionagent (lipid or polycationic polymer) where the peptide or protein ormodified peptide or protein is non-covalently associated with thenucleic acid. Without being bound by any theory, applicants believe thatthe complexes of the present invention are lipid aggregates thattypically contain liposomal or lipid aggregate structures, although theprecise nature of these structures is not presently known. Accordingly,in certain illustrative examples, complexes of the present invention areliposomal complexes. The entire complex, or a portion of the complex,such as a lipid portion, for example a lipid of Formula I, can beformulated into liposomes, for example using the method of reverseevaporation, which is well known in the art. Alternatively the lipidportion of the complex or the entire complex, can be formulated by otherwell-known methods for liposome formation such as sonication ormicrofluidization. These liposome formulations are effective fortransfecting DNA into cultured cells.

In one embodiment, a complex containing the non-naturally occurringpeptide- or protein of the invention and the nucleic acid (where thenon-naturally occurring peptide or protein can optionally be conjugatedto a nucleic-acid binding group) is first formed and then combined witha cationic lipid, such as a lipid of Formula I, for transfection. In arelated embodiment, a peptide- or protein-lipid conjugate is combinedoptionally with other lipids, including any appropriate cationic lipid,and then combined with nucleic acid for transfection. In another relatedembodiment, a nucleic acid-lipid complex is formed and then combinedwith a non-naturally occurring peptide or protein for transfection. Asdiscussed above, the lipid-containing complexes of any of theseembodiments can be liposomal or non-liposomal formulations. Furthermore,any of the complexes formed in these embodiments can be stored, forexample, for 5 minutes to 1 year, or for 15 minutes to 6 months, or for1 hour to 3 months, before transfecting cells. In the case of a peptideor protein-lipid conjugate, such a conjugate can be stored for example,for 5 minutes to 1 year, or for 15 minutes to 6 months, or for 1 hour to3 months, before combining with nucleic acid.

In another embodiment, a complex containing the non-naturally occurringpeptide or protein and the nucleic acid (where the non-naturallyoccurring peptide or protein can be conjugated to a nucleic-acid bindinggroup) is formed and then combined with a polycationic polymer fortransfection. In a related embodiment, a peptide-polycationic polymerconjugate is combined optionally with another polycationic polymer andthen combined with nucleic acid for transfection. In another relatedembodiment, a nucleic acid-polycationic polymer complex is formed andthen combined with a peptide or protein for transfection. A polycationicpolymer and/or peptide-conjugated polycationic polymer can be combinedwith cationic lipids and cationic lipid composition to obtain improvednucleic acid transfection compositions. In accordance with theinvention, multiple peptides and/or proteins may be added to accomplishtransfection.

Transfection compositions of this invention comprising peptide- orprotein-lipid conjugates and nucleic acids can further include othernon-peptide or non-protein agents that are known to further enhancetransfection.

Transfection compositions of this invention comprising peptide- orprotein-polycationic polymer conjugates and nucleic acid can furtherinclude other non-peptide agents that are known to further enhancepolycationic polymer transfection, for example polycationic polymertransfection can be enhanced by addition of DEAE-dextran and/orchloroquine.

In one preferred though non-limiting embodiment, the non-naturallyoccurring peptide of the present invention may be first bound bynon-covalent association to a nucleic acid or other cargo to beintroduced into a cell. The peptide-nucleic acid complexes are thenadmixed with a transfection agent (or mixture of agents) and theresulting mixture is employed to transfect cells. Preferred transfectionagents are cationic lipid compositions, such as but not limited to thosecontaining a lipid of Formula (I), particularly monovalent andpolyvalent cationic lipid compositions, more particularly cationic lipidcompositions composed of a 1:1 to 4:1 mixtures of cationic lipid andDOPE and a 1:1 to 4:1 mixtures of cationic lipid and cholesterol, aswell as a a 1:1 to 4:1 mixtures of cationic lipid and DOPC, moreparticularly cationic lipid compositions composed of a 1:1 to 4:1mixtures of dihydroxyl-dimyristylspermine tetrahydrochloride and DOPEand a 1:1 to 4:1 mixtures of dihydroxyl-dimyristylsperminetetrahydrochloride and cholesterol, as well as a a 1:1 to 4:1 mixturesof dihydroxyl-dimyristylspermine tetrahydrochloride and DOPC as well asa 1:1 to 4:1 mixtures of hydroxyl-dimyristylspermine tetrahydrochlorideand DOPE and a 1:1 to 4:1 mixture of hydroxyl-dimyristylsperminetetrahydrochloride and cholesterol, as well as a 1:1 to 4:1 mixtures ofhydroxyl-dimyristylspermine tetrahydrochloride and DOPC.

In another optional embodiment, a mixture of one or moretransfection-enhancing peptides, proteins, or protein fragments,including fusogenic peptides or proteins, transport or traffickingpeptides or proteins, receptor-ligand peptides or proteins, or nuclearlocalization peptides or proteins and/or their modified analogs (e.g.,spermine modified peptides or proteins) may be complexed with nucleicacid at the same time or immediately after complexation of the nucleicacid with the non-naturally occurring peptide of the present inventionto be introduced into a cell. The peptide-nucleic acid complexes arethen admixed with transfection agent and the resulting mixture isemployed to transfect cells. In certain embodiments, the mixture of thetransfection enhancing peptide, protein, or protein fragment is storedbefore it is complexed with nucleic acid.

In another optional embodiment, a component of a transfection agent(lipids, neutral lipids, helper lipids, cationic lipids, dendrimers, orPEI) may be covalently conjugated to selected peptides, proteins, orprotein fragments directly or via a linking or spacer group. Ofparticular interest in this embodiment are peptides or proteins that arenon-naturally occurring fusogenic proteins from non-enveloped virusessuch as are known in the art.

Exemplary Uses of the Complexes Containing Non-Naturally OccurringPeptides of Non-Enveloped Viruses

The delivery methods employing the lipid aggregates of the presentinvention or mixtures thereof can be applied to cells in vitro, ex vivo,and in vivo, particularly for transfection of eukaryotic cells ortissues including animal cells, human cells, non-human animal cells,insect cells, plant cells, avian cells, fish cells, mammalian cells andthe like. The polyanion that is to be delivered into the cell iscontacted with lipid aggregates in the presence of a non-naturallyoccurring peptide as described above to form apolyanion-lipid-polypeptide aggregate complex. The target cell or cellsare then incubated with the complex, or, for in vivo applications, thecomplex is administered to the organism so that the complex contacts thetarget cells or tissue. The compounds of the present invention may alsobe conjugated to or mixed with or used in conjunction with a variety ofuseful molecules and substances, also referred to as transfectionhelpers, such as proteins, peptides, growth factors and the like toenhance cell-targeting, uptake, internalization, nuclear targeting andexpression.

The complexes and methods of the present invention, especially thoseinvolving transfection compositions that include complexes providedherein, can be used for in vitro and in vivo transfection of cells,particularly of eukaryotic cells, and more particularly to transfectionof higher eukaryotic cells, including animal cells. The methods of thisinvention can be used to generate transfected cells which express usefulgene products. The methods of this invention can also be employed as astep in the production of transgenic animals. The methods of thisinvention can be useful as a step in any therapeutic method requiringintroduction of nucleic acids into cells including methods of genetherapy and viral inhibition and for introduction of antisense orantigene nucleic acids, ribozymes, RNA regulatory sequences, siRNA,RNAi, Stealth® RNAi (Invitrogen Corporation, Carlsbad Calif.) or relatedinhibitory or regulatory nucleic acids into cells. In particular, thesemethods may be useful in cancer treatment, in in vivo and ex vivo genetherapy, and in diagnostic methods.

The transfection compositions and methods of this invention comprisingpeptides, proteins, peptide or protein fragments or modified peptides ormodified proteins, can also be employed as research agents in anytransfection of eukaryotic cells done for research purposes.

Accordingly, provided herein is a method of introducing a macromoleculeinto a cell, that includes forming a transfection composition thatincludes a nucleic acid and a complex comprising a transfection agentand a fusion agent, wherein the fusion agent includes a fusion promotingamino acid sequence derived from a fusion protein of a non-envelopedvirus; and contacting a eukaryotic cell with the transfectioncomposition. Provided in the Examples section herein are illustrativeprotocols for using compositions of the present invention to transfecteukaryotic cells. As disclosed herein, the fusion agent in illustrativeexamples is a membrane fusion peptide (MPP), advantageously a fusionpeptide that is between 5 and 50 amino acids in length where at least 5contiguous amino acids of the fusion peptide are at least 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 100% similar to any of the peptides setforth in Table 1.

A further embodiment provides a method of transfecting a cell or tissuewith a nucleic acid in vivo wherein the method comprises forming a lipidaggregate, preferably a liposome, comprising one or more cationiclipids, optionally one or more neutral lipids and optionally one or morehelper lipids, contacting the lipid aggregate with the nucleicacid-peptide complex formed by contacting the nucleic acid to anon-naturally occurring peptide of the present invention underconditions sufficient to promote the stable non-covalent interactionbetween the peptide and the nucleic acid, thereby forming a neutral orpositively charged lipid aggregate-nucleic acid complex, andadministering the lipid aggregate-nucleic acid complex to the organismso that the complex contacts the target cells or tissue.

Administration of the lipid aggregate-peptide-nucleic acid complex canbe achieved orally, intravenously, or by subcutaneous or intramuscularinjection or applied topically to the tissue or to cells in culture in alaboratory setting.

Optionally, the polyanion-peptide-lipid aggregate complex is stored fora period prior to being contacted with the cell or cells fortransfection. The polyanion-peptide-lipid aggregate complex is stableand can be stored for a time period of at least 30 minutes, at least 45minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4hours, at least 5 hours, at least 10 hours, at least 15 hours, at least20 hours, at least 24 hours, at least 48 hours, at least 72 hours, atleast 5 days, at least 7 days, at least 14 days, at least 28 days, atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months or at least 1 year, or for a timeperiod between any of these time periods.

In another embodiment, lipid aggregates of the present invention(approximately between 1 μl and 2000 μl) are provided in the wells of amultiwell plate. Target polyanion molecules to be delivered into targetcells are selected and added to the wells to formpolyanion-peptide-lipid aggregate complexes, which are subsequentlycontacted with the target cells. The lipid aggregates can have the samecomposition and concentration in each well, or the lipid aggregatecomposition and/or concentration can vary from well to well. Where thepolyanions are nucleic acids such as DNA or RNA, the nucleic acids canbe added to the wells and optionally stored before contacting with thetarget cells.

The methods of this invention optionally comprise the step of contactingthe one or more cationic lipids with one or more helper or neutrallipids before or at the same time as contacting the nucleic acid-peptidecomplex with the one or more cationic lipids to form lipid aggregatesencapsulating the nucleic acid-peptide. The methods also optionallycomprise forming the lipid aggregates into liposomes prior to contactwith the nucleic acid. In further embodiments, the liposomes are formedby microfluidization, extrusion or other means known in the art. Thenucleic acids are preferably DNA or RNA that inhibit expression of atarget gene. Preferably the nucleic acid associates with a transcript ofthe gene to effect inhibition. Preferably, the nucleic acid is RNAi,siRNA, shRNA, or stRNA, and is optionally chemically modified.

Volumes and concentrations of nucleic acid or other macromolecule,volume and concentration of the transfection complexes provided herein,volumes and compositions of diluents, and volume and concentration ofcells, can be determined using standard experimental approaches for suchoptimization and titration, including, for example, methods that utilizecytotoxicity assays and/or methods that employ transfection usingnucleic acid expression vectors that express reporter genes, such asbeta galactosidase, luciferase, and/or fluorescent proteins.Furthermore, cell densities can be optimized using standard methods, andcell densities for transfections using the transfection complexesprovided herein can range, for example, from high density>75% to lowdensity<50%

Exemplary diluents for complexation reactions, for example, includereduced-serum, or serum-free media, such as D-MEM and RPMI 1640 andOptiPro™, Opti-MEM® (Invitrogen Corporation). Incubation times forforming complexes can be determined using routine methods, althoughtypical incubation times are between 5 and 240 minutes. In addition, itwill be understood that media for culturing of cells before and aftertransfection can be chosen based on the cell line to be transfected andbased on the particular application of the method. For example, for theproduction of proteins in suspension cells, in illustrative embodiments,reduced serum, or advantageously serum-free, medium can be used. Incertain illustrative embodiments, animal origin-free medium is employed,such as, but not limited to, 293 Expression Medium (InvitrogenCorporation) and CD-CHO Medium (Invitrogen Corporation). In certainaspects depending on the cell type to be transfected, antibiotics can beexcluded from post-transfection media. Incubation times forpost-transfection culturing of cells varies depending on the cell typeand the desired outcome of the transfection, but typically ranges from 2hours to 7 days. For large-scale protein production, cells can beincubated, as a non-limiting example, for between 1 day and 7 days.

It will be understood that a wide range of concentrations oftransfection agent and a fusion agent can be used in the complexes,compositions and methods provided herein. For example, in anillustrative non-limiting example of a composition that includes acomplex of a cationic lipid and a non-naturally occurring peptide, thetotal exemplary, non-limiting combined concentration of cationic lipidand non-naturally occurring peptide in the composition can be between 1mg/ml and 4 mg/ml. The range of peptide added to the lipid at 1 mg/mlcan between 100 μg/ml and 3 mg/ml. The ratio of the cationic lipid tohelper lipid can between 0.5/1.0 (molar) and pure compound.

Cells that can be transfected according to the present inventioninclude, for example, virtually any eukaryotic cell including primarycells, cells in culture, and cells in cultured tissue, particularly cellthat are considered difficult to transfect. The cells can be attachedcells or cells in suspensions. In certain illustrative aspects, thecells are suspension CHO-S cells and suspension 293-F cells. Suspensioncell cultures are particularly well-suited for protein productionmethods provided herein. Other cells that can be transfected using theagents and methods of the invention include, but are not limited to,293, such as GripTite 293 MSR (Invitrogen Corporation), CHO, Cos7,NIH3T3, Hela, primary fibroblast, A549, Be2C, SW480, Caco2, primaryneurons, Jurkat, C6, THP1, IMR90, HeLa, ChoK1, GT293, MCF7, HT1080,LnCap, HepG2, PC12, SKBR3, and K562 cells, or any cells listed in Table6.

In certain embodiments provided herein, a transfection enhancing agentis included in the complex that is used to transfect cells. For examplethe transfection enhancing agent can be a nuclear localization peptide.In one example, the transfection enhancing agent is the PLUS™ Reagent(Invitrogen Corporation). It has been shown that the addition of PLUS™reagent enhances protein expression when used together with transfectioncompositions as provided herein. Cytotoxicity was not affected by theuse of the PLUS™ Reagent.

In another embodiment, provided herein is a method for producing aprotein comprising, transfecting a cell with a nucleic acid moleculeencoding the protein, incubating the cell to produce the protein, andcollecting the protein, wherein the transfecting is performed bycontacting the cell with a transfection composition including anon-naturally occurring peptide of the present invention. Thecomposition for transfecting the cell can be any compositions asprovided herein. Exemplary compositions include the nucleic acidmolecule encoding the protein of interest, complexed with anon-naturally occurring peptide of the present invention, optionally afusion agent, and a transfection agent.

In illustrative embodiments the encoded protein is an antibody molecule,or an antigen binding fragment or derivative portion thereof, forexample a single chain Fv fragment. In these embodiments, the method canfurther include isolating the protein, for example, by using affinitypurification on an antibody-binding column. In certain examples, nucleicacids encoding both chains of an antibody are transfected into cellsusing a transfection composition provided herein.

It will be understood that the nucleic acid encoding the protein can bean expression vector. The expression vector typically has a promoteroperatively linked to one or more nucleic acid sequences encoding one ormore protein chains. Where the protein produced is a pharmaceuticalproduct, the protein can be formulated accordingly, for example in anappropriate choice of physiologic medium.

The transfection composition provided herein can also be used tointroduce peptides and proteins and the like into cells using methodsthat are known in the art. Methods of using cationic lipids for peptideand protein delivery previously have been described. In addition, thetransfection compositions can be used to deliver nucleic acids, peptidesand proteins and the like into tissues in vivo. Methods of using lipidsfor delivering compounds to tissue in vivo previously have beendescribed. The transfection compositions can, with appropriate choice ofphysiologic medium, be employed in therapeutic and diagnosticapplications.

Reagent Kits:

The invention is further directed to kits containing, in at least afirst suitable container, at least one non-naturally occurring peptidein accordance with the present invention. The kits of the presentinvention can further comprise one or more containers comprising areagent that facilitates the introduction of at least one macromolecule,e.g., a cationic transfection reagent, optionally one or more helperlipids or neutral lipids, and may optionally be provided with a cargo,such as, e.g., a nucleic acid or other cargo as defined above. Preferredtransfection reagents include, but are not limited to, cationic lipidsand the like.

Components of the transfection compositions of this invention can beprovided in a reagent kit. The kit may contain a transfection agent anda non-naturally occurring peptide of the present invention. This kit canalso optionally include a transfection enhancing agent such as atransfection-enhancing peptide, protein or fragment thereof or atransfection enhancing compound. The transfection agent, thenon-naturally occurring peptide, and the optional transfection enhancingagent, when present, can each be included as a mixture (i.e. in a singlecontainer, typically a tube and/or vial), or can be included as separateportions (i.e. in separate containers, for example separate vials and/ortubes). The kits of the present invention, as will be understood,typically include vessels, such as vials and/or tubes, which arepackaged together, for example in a cardboard box or other packaging.The kits can be shipped from a supplier to a customer. For example, inone example provided herein is a kit that includes a vial that includesa liposomal formulation that includes a transfection agent and atransfection enhancing peptide. The kit can also include, for example, aseparate vessel that includes a transfection enhancing agent, such as atransfection enhancing peptide, for example Plus Reagent™ (InvitrogenCorp., Carlsbad, Calif.). The kit can also include in separatecontainers, cells, cell culture medium, and a reporter nucleic acidsequence, such as a plasmid that expresses a reporter gene. In certainexamples, the culture medium can be reduced-serum medium and/or proteinexpression medium.

In one embodiment, a kit comprises individual portions of, or a mixtureof, cationic lipid, such as but not limited to a lipid of Formula I,optionally in combination with one or more helper lipids and/or one ormore neutrallipids, and peptide, protein or fragment thereof or modifiedpeptide, protein or fragment thereof of the present invention. Inanother embodiment, a kit comprises individual portions of, or a mixtureof, polycationic polymers and peptide, protein or fragments thereof ormodified peptide, protein or fragments thereof of the present invention.Cationic lipid transfection kits can optionally include neutral lipid aswell as other transfection-enhancing agents or other additives, and therelative amounts of components in the kit may be adjusted to facilitatepreparation of transfection compositions. Kit components can includeappropriate medium or solvents for other kit components.

Cationic lipid transfection kits comprising a monocationic orpolycationic lipid composition, such as but not limited to a lipid ofFormula I, and further including a neutral lipid and a peptide orprotein of the present invention are preferred.

Dendrimer transfection kits can optionally include other transfectionenhancing agents, such as DEAE-dextran and/or chloroquine, as well asother additives and the relative amounts of components in the kit may beadjusted to facilitate preparation of transfection compositions.

Kits provided by this invention include those comprising an individualportion of a polycationic lipid composition comprising DOSPA and DOPE ora monocationic lipid composition comprising DOTMA and DOPE and a portionof modified peptide, optionally a spermine- or spermidine-modifiedpeptide. Kits provided by this invention include those comprising anindividual portion of a polycationic polymer and a portion of aspermine-modified peptide.

In related embodiments, kits of this invention can comprise a peptide-or protein-lipid conjugate or a peptide- or protein-polycationic polymerconjugate in combination with non-conjugated lipids, non-conjugatedpolycationic polymer and other agents to facilitate transfection.

Kits of this invention can include those useful in diagnostic methods,e.g., diagnostic kits which in addition to transfection agent andtransfection-enhancing agents (e.g., proteins, peptides, and fragmentsand modifications of peptides and proteins) can contain a diagnosticnucleic acid. A diagnostic nucleic acid is a general term for anynucleic acid which can be employed to detect the presence of anothersubstance (most generally an analyte) in a cell. For example, whentransfected into a cell a diagnostic nucleic acid may increase ordecrease expression of a gene therein in response to the presence ofanother substance in the cell (e.g., a protein, small molecule, steroid,hormone, or another nucleic acid). Diagnostic nucleic acids also includethose nucleic acids that carry some label or otherwise detectable markerto a particular target cell or target tissue for detection of the targetcell or tissue or for detection of a substance in the target cell ortissue.

Nucleic acids that can be transfected by the methods of this inventioninclude DNA and RNA of any size from any source comprising natural basesor non-natural bases, and include those encoding and capable ofexpressing therapeutic or otherwise useful proteins in cells, thosewhich inhibit undesired expression of nucleic acids in cells, thosewhich inhibit undesired enzymatic activity or activate desired enzymes,those which catalyze reactions (ribozymes), and those which function indiagnostic assays (e.g., diagnostic nucleic acids). Therapeutic nucleicacids include those nucleic acids that encode or can expresstherapeutically useful proteins, peptides or polypeptides in cells,those which inhibit undesired expression of nucleic acids in cells, andthose which inhibit undesired enzymatic activity or activate desiredenzymes in cells.

The compositions and methods provided herein can also be readily adaptedin view of the disclosure herein to introduce biologically-activemacromolecules other than nucleic acids including, among others,polyamines, polyamine acids, polypeptides and proteins into eukaryoticcells. Other materials useful, for example as therapeutic agents,diagnostic materials, research reagents, which can be bound to thepeptides and modified peptides and introduced into eukaryotic cells bythe methods of this invention.

The lipids of Formula I can be used as the cationic lipid(s) of the kitsdescribed above, and may independently be provided in a reagent kit. Ingeneral, the kit contains a lipid of Formula (I) in a suitablecontainer. The lipid may be, for example, in a solution of an organicsolvent, such as ethanol, in a buffer, or in a solvent/buffer mixture Inaddition, the kit may include, but is not limited to, a lipid of Formula(I), and an amino acid sequence from a non-naturally occurring proteinthat enhances or promotes membrane fusion of a liposome carrier with acell membrane in a suitable solvent or buffer.

In one embodiment, a kit may comprise individual portions of, or amixture of, e.g., lipids of Formula (I) or other cationic lipids andpeptide, protein or fragment thereof or modified peptide, protein orfragment thereof. Kits which include lipids of Formula (I) or othercationic lipids can optionally include neutral lipid as well as othertransfection-enhancing agents or other additives, and the relativeamounts of components in the kit may be adjusted to facilitatepreparation of transfection compositions. Kit components can includeappropriate medium or solvents for other kit components.

Kits which include lipids of Formula (I) or other cationic lipids, aneutral lipid and a modified peptide or protein are preferred. Kitsprovided by this invention include those composition comprising anindividual portion of a lipid of Formula (I), DOPE and a portion ofpeptide, particularly a spermine-modified peptide. Kits provided by thisinvention include those comprising an individual portion of a lipid ofFormula (I), and a portion of a modified peptide containing a stretch ofbasic amino acids such lysine, ornithine, or arginine.

Methods for Selling

Also provided is a method for selling a non-naturally occurring peptide,lipid, transfection complex, transfection composition, and/or kitprovided herein, comprising presenting to a customer an identifier thatidentifies the non-naturally occurring peptide, lipid, complex and/ortransfection composition, and/or a kit provided herein, and providingaccess to the customer to a purchase function for purchasing thenon-naturally occurring peptide, lipid, transfection complex,transfection composition, and/or kit provided herein using theidentifier. The identifier is typically presented to the customer aspart of an ordering system. The ordering system can include an inputfunction for identifying a desired product, and a purchasing functionfor purchasing a desired product that is identified. The ordering systemis typically under the direct or indirect control of a provider. Acustomer as used herein, refers to any individual, institution,corporation, university, or organization seeking to obtain biologicalresearch products and services. A provider as used herein, refers to anyindividual, institution, corporation, university, or organizationseeking to provide biological research products and services.

The present invention also provides a method for selling a non-naturallyoccurring peptide, lipid, transfection complex, transfectioncomposition, and/or kit provided herein, comprising: presenting to acustomer an input function of a telephonic ordering system, and/orpresenting to a customer a data entry field or selectable list ofentries as part of a computer system, wherein the non-naturallyoccurring peptide, lipid, transfection complex, transfection compositionand/or kit is identified using the input function. Where the inputfunction is part of a computer system, such as displayed on one or morepages of an Internet site, the customer is typically presented with anon-line purchasing function, such as an online shopping cart, whereinthe purchasing function is used by the customer to purchase theidentified non-naturally occurring peptide, lipid, transfection complex,transfection composition, and/or kit. In one aspect, a plurality ofidentifiers are provided to a customer, each identifying a differentnon-naturally occurring peptide, lipid, complex and/or transfectioncomposition, and/or a kit provided herein, or a different volume orweight of the non-naturally occurring peptide, lipid, complex and/ortransfection composition, and/or a kit provided herein. The method mayfurther comprise activating the purchasing function to purchase thelipid, transfection complex, transfection composition, and/or kitprovided hererin. The method may still further comprise shipping thepurchased non-naturally occurring peptide, lipid, transfection complex,transfection composition, and/or kit provided herein to the customer.The non-naturally occurring peptide, lipid, transfection complex,transfection composition, and/or kit can be shipped by a provider to thecustomer. The provider typically controls the input function, and cancontrol the web site accessed to access the input function to purchase anon-naturally occurring peptide, lipid, complex and/or transfectioncomposition, and/or a kit provided herein.

Pharmaceutical Compositions

Transfection agents and transfection-enhancing agents of this inventioncan be provided in a variety of pharmaceutical compositions and dosageforms for therapeutic applications. For example, injectableformulations, intranasal formulations and formulations for intravenousand/or intralesional administration containing these complexes can beused therapy.

In general the pharmaceutical compositions of this invention shouldcontain sufficient transfection agent and any enhancing agents (peptide,protein, etc.) to provide for introduction of a sufficiently high enoughlevel of nucleic acid into the target cell or target tissue such thatthe nucleic acid has the desired therapeutic effect therein. The levelof nucleic acid in the target cell or tissue that will betherapeutically effective will depend on the efficiency of inhibition orother biological function and on the number of sites the nucleic acidmust affect.

The dosage of transfection compositions described herein administered toa patient will depend on a number of other factors including the methodand site of administration, patient age, weight and condition. Those ofordinary skill in the art can readily adjust dosages for a given type ofadministration, a given patient and for a given therapeutic application.

It will be appreciated by those of ordinary skill in the art that thetransfection composition should contain minimal amounts of inhibitorycomponents, such as serum or high salt levels, which may inhibitintroduction of nucleic acid into the cell, or otherwise interfere withtransfection or nucleic acid complexation. It will also be appreciatedthat any pharmaceutical or therapeutic compositions, dependent upon theparticular application, should contain minimal amounts of componentsthat might cause detrimental side-effects in a patient.

The transfection compositions described herein may be formulated intocompositions which include a pharmaceutically active agent and apharmaceutically acceptable diluents, excipients or carriers therefor.Such compositions may be in unit dosage forms such as tablets, pills,capsules (including sustained-release or delayed-release formulations),powders, granules, elixirs, tinctures, syrups and emulsions, sterileparenteral solutions or suspensions, aerosol or liquid sprays, drops,ampoules, auto-injector devices or suppositories; for oral, parenteral(e.g., intravenous, intramuscular or subcutaneous), intranasal,sublingual or rectal administration, or for administration by inhalationor insufflation, and may be formulated in an appropriate manner and inaccordance with accepted practices such as those disclosed inRemington's Pharmaceutical Sciences, (Gennaro, ed., Mack Publishing Co.,Easton Pa., 1990, herein incorporated by reference).

Some examples of suitable carriers, excipients and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, methyl cellulose, methyl- and propyl-hydroxybenzoates, talc,magnesium stearate and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents or flavoring agents. Whenthe carrier serves as a diluent, it may be a solid, semi-solid or liquidmaterial which acts as a vehicle, excipient or medium for the activeingredient. In the case of injections, it is possible to preparesolutions or liposomes of one or more lipids of the present invention inpharmaceutically acceptable carriers such as an aqueous or nonaqueoussolvent. Examples of solvents which may be used are distilled water forinjection, physiological saline solution, Ringer's solution, plant oil,synthetic fatty acid glycerides, higher fatty acid esters, propyleneglycol, and the like.

EXAMPLES

The following Examples are provided to illustrate certain aspects of thedisclosure and to aid those of skill in the art in practicing thedisclosure. These Examples are in no way to be considered to limit thescope of the disclosure in any manner.

Example 1. Preparation of Lipid Aggregate/Polypeptide Complexes andTransfection of Cultured Cells

All cell were cultured under standard culture conditions recommended foreach cell line by the American Type Culture Collection (ATCC).Approximately 24 hrs prior to transfections, cells were seeded so thatthey would be 70-90% confluent on the day of transfection. The followingguidelines are generally applicable, though minor variations exist aswill be readily appreciated by one skilled in the art, and depending onthe identity of the cell line, its growth characteristics and needs, andits morphology in adherent culture. Generally, for a 96-well plate,between 1-4×10⁴ cells per well were seeded, for a 24-well plate0.5-2×10⁵ cells per well were seeded, for a 6-well plate 0.25-1×10⁶cells were seeded.

Transfection complexes for transfecting DNA into cells in culture wereprepared according to manufacturer protocol. LIPOFECTAMINE® 2000 andLIPIFECTAMINE® LTX were purchased from Life Technologies Corp.(Carlsbad, Calif.), FUGENE® HD was purchased from Promega Corp.(Fitchburg, Wis.), and X-TREMEGENE™ HP was purchased from RocheDiagnostics (Basel, Switzerland).

To prepare transfection complexes containing the non-naturally occurringpeptides described above, Peptide 1 having the following sequenceSRRARRSPRESGKKRKRKRGGGSGGGSGGGSRRRRRRRRRRR (SEQ ID NO. 89) wassynthesized and provided as a dry powder. The dry powder wasreconstituted in sterile ultrapure water to a final concentration of4.35 mg/ml and allowed to fully dissolve. This stock peptide solutionwas set aside for use in the next step.

To prepare the lipid aggregate-DNA-peptide complexes, LIPOFECTAMINE®3000 reagent (Life Technologies Corp., Carlsbad, Calif.) was obtained.For each well of a 96-, 24- or 6-well plate of cells to be transfected a5 μl, 25 μl, and 125 μl, aliquot of Gibco® Opti-MEM® medium was placedin separate disposable plastic Eppendorf tube. Between 0.1 μl to 0.6 μlfor a 96-well plate, 0.5 μl to 3.0 μl for a 24-well plate, or 2.0 μl to15 μl for a 6-well plate of LIPOFECTAMINE® 3000 reagent was added to thealiquoted Opti-MEM®, mixed well and incubated at room temperature.

In a separate Eppendorf tube, 5 μl (for each well of a 96-well plate),25 μl (for each well of a 24-well plate), or 125 μl (for each well of a6-well plate) of Gibco® Opti-MEM® medium was aliquoted into a tube foreach well of cultured cells to be transfected, into which was mixed 0.1m of pcDNAEF1a/emGFP or GST-STAT expression vector DNA for each well ofa 96-well plate, 0.5 μg of pcDNAEF1a/emGFP or GST-STAT expression vectorDNA for each well of a 24-well plate, and 2.5 μg of pcDNAEF1a/emGFP orGST-STAT expression vector DNA for each well of a 6-well plate.

Into the diluted DNA mixture was added 0.2 μl of the stock peptide foreach well of a 96-well plate, 1 μl of stock peptide for each well of a24-well plate, and 5 μl of the stock peptide solution for each well of a6-well plate, and the peptide/DNA mixture was mixed well and incubatedfor approximately 1 minute at room temperature.

For each well of a 96-well plate, 5 μl of the diluted DNA/peptidemixture was mixed with 5 μl of the diluted LIPOFECTAMINE® 3000 reagent,for each well of a 24-well plate, 25 μl of the diluted DNA/peptidemixture was mixed with 25 μl of the diluted LIPOFECTAMINE® 3000 reagent,and for each well of a 6-well plate, 125 μl of the diluted DNA/peptidemixture was mixed with 125 μl of the diluted LIPOFECTAMINE® 3000reagent, and lipid-peptide-DNA complexes were allowed to form byincubating the resulting mixture for approximately 5 minutes at roomtemperature.

Following the incubation, the lipid-peptide-DNA complexes were added tocells that were seeded the previous day with fresh growth medium; for96-well plates, 10 μl of the lipid-peptide-DNA was added to the cells,for 24-well plates, 50 μl of the lipid-peptide-DNA mixture was added tothe cells, for 6-well plates, 250 μl of the lipid-peptide-DNA was addedto the cells. The cells were incubated in the presence of thelipid-peptide-DNA for approximately 24-48 hrs, and analyzed.

Example 2. Transfection of Various Cell Lines

A panel of 10 difficult-to-transfect cancer cell lines (HepG2, Hepa1-6,Hep3B, HUH7, MCF-7, MDA-MB-23, SKBR3, LNCaP, Bend3, and T986), twodifficult to transfect neuronal cell lines (PC12 and Neuro2A), twodifficult to transfect myoblast cell lines (H9C2 and C2C12) and adifficult to transfect kidney fibroblast cell line (Vero) weretransfected with pcDNAEF1a/emGFP, an expression vector encoding GFP,according to the methods set forth in Example 1. After 24 hrs in thepresence of the transfection complexes, the cells were visualized usingfluorescence microscopy at the appropriate wavelength.

Expression of GFP in the cancer cell lines is shown in FIG. 1A, inneuronal cells is shown in FIG. 1B, in Myoblast cells is shown in FIG.1C, and in kidney fibroblast cells is shown in FIG. 1D.

Example 3. Comparison of Various Transfection Reagents

A panel of six different cell lines (HEK293, HeLa, COS-7, LNCaP, A549and HepG2) were transfected with pcDNAEF1a/emGFP using FUGENE® HD,LIPOFECTAMINE® 2000, or LIPOFECTAMINE® 3000 in combination with Peptide1 as described in Example 1. The cells were allowed to transfect for 48hrs. The results shown in FIG. 2 show that, while both FUGENE® HD andLIPOFECTAMINR® 2000 were able to transfect a small portion of each ofthe cells (FIG. 2, first two columns), the presence of Peptide 1 in thetransfection complex improved transfection efficiency substantially(FIG. 2, last column).

To extend this study, a panel of 61 different cell lines weretransfected as above using either LIPOFECTAMINE® 2000 or LIPOFECTAMINE®3000 in combination with Peptide 1 as described in Example 1.Approximately 48 hours after transfection, transfected cells wereexamined by fluorescence microscopy to determine relative transfectionefficiency, and cellular extracts were prepared and analyzed using aFL600 Fluorescence Microplate Reader to measure fold improvement in GFPexpression of LIPOFECTAMINE® 3000 with Peptide 1 over LIPOFECTAMINE®2000. The results are shown in Table 6. As can be seen, useLIPOFECTAMINE® 3000 reagent in combination with Peptide 1 yields highertransfection efficiencies and protein expression than LIPOFECTAMINE®2000 reagent when tested in a variety of cell lines.

TABLE 6 Performance of LIPOFECTAMINE ® 3000 and Peptide1 lipid aggregateformulations in vitro in various cell lines as measured by relativetransfection efficiency and fold improvement of transfection efficiencycompared to LIPOFECTAMINE ® 2000 transfection. Fold improvement ofRelative protein expression over transfection LIPOFECTAMINE ® Cell lineCell/Tissue lineage efficiency (%) 2000 3T3 Mouse embryonic fibroblast,51-79% 11 immortalized 4T1 Mouse breast tumor, epithelial 51-79% 2 A431Human epidermoid  <30% 4 carcinoma, epithelial A549 Human lungcarcinoma, 51-79% 3 epithelial ACHN Human metastatic kidney cell, 30-50%2 adenocarcinoma bEnd.3 Mouse brain endothelioma,  <30% 9 viraltransformed BJ Human foreskin, immortalized  <30% 3 epithelial BT-549Human breast carcinoma, 51-79% 4 epithelial C2C12 Mouse myoblast, 51-79%14 immortalized C6 Rat glioma 30-50% 5 Caco-2 Human colorectalcarcinoma, 51-79% 2 epithelial Caki-1 Human kidney carcinoma,  <30% 4epithelial CHO-K1 Chinese hamster ovary, 51-79% 1 immortalizedepithelial CHO-S Chinese hamster ovary,  <30% 1 suspension adapted COLO205 Human colorectal carcinoma,  <30% 4 epithelial COS-7 African greenmonkey kidney 51-79% 4 fibroblast, virus transformed DU 145 Humanmetastatic prostate 30-50% 2 tumor, epithelial H460 Human lungcarcinoma, large 51-79% 3 cell, epithelial H9c2 Rat embryonic myoblast51-79% 3 (heart) HCC1937 Human mammary tumor,  <30% 5 epithelial HCT116human colon carcinoma,  >80% 1 epithelial HEK 293 Human embryonic kidney >80% 2 fibroblasts, immortalized HeLa Human cervical carcinoma,  >80% 3epithelial Hep-3B Human hepatocellular 51-79% 2 carcinoma, epithelialHepa 1-6 Mouse hepatocellular 51-79% 6 carcinoma, epithelial HepG2 Humanhepatocellular  >80% 16 carcinoma, epithelial Hs 578T Human breastcarcinoma,  >80% 3 epithelial cHT29 human colon carcinoma,  <30% 1epithelial Huh-7 Human hepatocellular 51-79% 4 carcinoma, epithelialJurkat Human T cell, immortalized  <30% 1 K-562 Human myelogenous 30-50%2 leukemia L6 Rat myoblast 30-50% 8 L929 Mouse fibrosarcoma Up to 30%  2 LNCaP Human prostate  >80% 10 adenocarcinoma MCF 10A Human breastcarcinoma, 30-50% 5 epithelial MCF7 Human breast carcinoma, 30-50% 2epithelial MDA-MB-231 Human breast carcinoma, 51-79% 3 epithelialMDA-MB-435 Human breast carcinoma, 51-79% 3 epithelial MDA-MB-468 Humanbreast carcinoma,  <30% 9 epithelial MDCK Canine kidney, immortalized <30% 1 Neuro-2a Mouse neuroblastoma  >80% 1 NCI-H23 Human lungadenocarcinoma 51-79% 2 NCI-H460 Human lung carcinoma, large  <30% 17cell P19 Mouse embryonal 30-50% 1 carcinoma/teratocarcinoma PANC-1 Humanpancreatic carcinoma, 51-79% 3 epithelial PC12 Rat pheochromocytoma51-79% 2 RAW264.7 Mouse macrophage, virus  <30% 4 transformed RBL-2H3Rat basophil leukemia  <30% 2 RD Human rhabdomyosarcoma 51-79% 4 Saos-2Human osteosarcoma 51-79% 4 SH-SY5Y Human neuroblastoma  <30% 1 SK-BR-3Human breast carcinoma, 51-79% 4 epithelial SK-MEL-28 Human melanoma51-79% 2 SK-N-SH Neuroblastoma cell line 30-50% 6 SK-OV-3 Human ovariancarcinoma 30-50% 3 SW480 Human colorectal 51-79% 2 adenocarcinoma SW620Human colorectal  <30% 5 adenocarcinoma T98G Human glioblastoma 51-79% 4U2OS Human osteosarcoma  >80% 3 U937 Human histiocytic leukemia  <30% 2Vero African green monkey kidney, 30-50% 8 epithelial

Example 4. Effect of Transfection Reagent Dosage on TransfectionEfficiency and Protein Expression

HeLa cells were plated in 96-well plates and transfected withpcDNAEF1a/emGFP using 0.1 μl, 0.2 μl, 0.3 μl or 0.4 μl of LIPOFECTAMINE®2000, LIPOFECTAMINE® LTX, or LIPOFECTAMINE® 3000 reagent in combinationwith Peptide 1 as described in Example 1. Transfection efficiency andrelative protein expression as measured by relative luminescence wasdetermine for each condition. The results are shown in FIG. 3.

FIG. 3A is a graph comparing the relative transfection efficiency for anexpression vector encoding GFP transfected into cultured HeLa cellsusing increasing dosages of three different commercially available lipidaggregate formulations, LIPOFECTAMINE® 2000 (open circles),LIPOFECTAMINE® LTX (open squares), and LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (open triangles).The presence of Peptide 1 in the transfection complex improvestransfection efficiency of the cells over the entire range oftransfection reagent dosages tested. The improvement to transfectionefficiency is particular pronounce at the lowest tested dosage.

FIG. 3B is a graph comparing the intensity of GFP expression in HeLacells transfected with an expression vector encoding GFP usingincreasing dosage of three different commercially available lipidaggregate formulations, LIPOFECTAMINE® 2000 (open circles),LIPOFECTAMINE® LTX (open squares), and LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (open triangles).The presence of Peptide 1 in the transfection complex improves relativeexpression of GFP in the cells over the entire range of transfectionreagent dosages tested. The improvement in protein expression isparticular pronounce at the lowest tested dosage.

Example 5. Improvement in Protein Expression Compared to ThreeCommercially Available Transfection Reagents

HepG2 cells were transfected in 24-well plates with an expression vectorencoding a GST-STAT fusion protein using LIPOFECTAMINE® 2000, FUGENE®HD, X-TREMEGENE™ HP or LIPOFECTAMINE® 3000 in combination with Peptide 1as described in Example 1. Approximately 24 hrs after transfection, celllysates were prepared using NOVEX® Cell Extraction Buffer (LifeTechnologies, Carlsbad, Calif.) and the lysates were resolved bySDS-PAGE electrophoresis, transferred to PVDF membranes, immunoblottedwith an anti-GST HRP-labeled polyclonal antibody, and detected withPierce™ ECL Western Blotting Substrate (Pierce Biotechnology, Rockford,Ill.).

FIG. 4 is a Western blot comparing the relative expression levels of aGST-STAT fusion protein (upper panel) in HepG2 cells transfected with anexpression vector encoding a GST-STAT fusion protein using the followingcommercially available lipid aggregate formulations: LIPOFECTAMINE® 2000(first lane), LIPOFECTAMINE® 3000 in combination with a peptideaccording to one embodiment (second lane), FUGENE® HD (third lane), andX-TREMEGENE™ HP (last lane). The bottom panel shows a western blot ofendogenous β-actin to confirm equal loading of cytosolic extract in eachlane.

Example 6. Transfection of H9 Human Embryonic Stem Cell Line

H9 Human embryonic stem cell line were seeded at a density of 37500cells/well in each well of a 96-well plate and transfected with 50 μg,100 μg or 200 μg of pcDNAEF1a/emGFP using 0.1 μl to 0.6 μl per well ofeither LIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 in combination withPeptide 1 as described in Example 1. After 24 hrs transfection,transfection efficiency was determined.

FIG. 5A is a graph comparing relative transfection efficiency of the H9human embryonic stem cell line (37,500 cells per well of a 96 wellplate) transfected with increasing dose of a GFP expression vector (50μg, left panel; 100 μg center panel, and 200 μg right panel) and usingbetween 0.1 to 0.6 μl per well of either LIPOFECTAMINE® 2000 (opentriangles) or LIPOFECTAMINE® 3000 in combination with a peptideaccording to an embodiment;

FIG. 5B is a representative fluorescence image of GFP expression in H9cells cultured in 96 well plates transfected with 100 μg/well using 200μl of either LIPOFECTAMINE® 2000 (left panel, demonstrating 18%transfection efficiency of H9 cells) or LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (right panel,demonstrating 52% transfection efficiency of H9 cells).

Example 7. Genomic Modification of Cells Using CRISPR Nuclease VectorSystem

Plasmid design and preparation: GENEART® Precision TALs and GENEART®CRISPR Nuclease Vectors were designed using the Life TechnologiesGENEART® web design tool(lifetechnologies.com/us/en/home/life-science/cloning/gene-synthesis/geneart-precision-tals.html).The forward and reverse TALENs contain the FokI nuclease and target theAAVS1 safe harbor locus. The all-in-one CRISPR vector system contains aCas9 nuclease expression cassette and a guide RNA cloning cassette thattarget the AAVS1 safe harbor locus, combined with a downstream orangefluorescent protein (OFP) reporter. A negative control plasmid, PCDNA™3.3, was also used throughout the assay. The plasmids were transformedinto competent E. coli cells. Clones were analyzed and sequenced forspecificity and then purified using a PURELINK® HiPure Plasmid FilterMaxiprep Kit to ensure low endotoxin activity and high-quality DNA.

U2OS and HepG2 cells were cultured using GIBCO® DMEM, high-glucose, withGLUTAMAX™ Supplement and 10% fetal bovine serum for 4-5 passages afterthawing; cells were dissociated using TRYPLE™ Express dissociationenzyme and seeded in a 12-well plate at 2×10⁵ cells per well in 1 mLcomplete medium to ensure 70-90% confluence on the day of transfection.

Transfection with LIPOFECTAMINE® 3000 Reagent in combination withPeptide 1 and LIPOFECTAMINE® 2000 Reagent was compared in each celltype. For transfection with LIPOFECTAMINE® 3000 Reagent, in separatetubes, 1.5 μL, of LIPOFECTAMINE® 3000 Reagent and 1 μg of plasmid DNAwere each diluted in 50 μL OPTI-MEM® Reduced-Serum Medium; then 2 μLPeptide 1 (see Example 1) was added to the diluted DNA. The diluted DNAwith Peptide 1 was added to the diluted LIPOFECTAMINE® 3000 Reagent andincubated at room temperature for 5 minutes. Then 100 μL, of theresulting complex was added to cells in complete medium. The procedurewas the same for LIPOFECTAMINE® 2000 Reagent, except that the amount oftransfection reagent was increased to 3 μL and no Peptide 1 was added tothe diluted DNA before adding it to the diluted LIPOFECTAMINE® 2000Reagent. All downstream analysis was performed 72 hourspost-transfection.

OFP expression from the CRISPR vector was determined by flow cytometryand microscopy. An EVOS® FL Imaging System was used to acquire imageswith the RFP filter. Cells were then dissociated 72 hourspost-transfection with TRYPLE™ Express enzyme and analyzed using a BDACCURI™ C6 Flow Cytometer with an FL-2 filter and blue laser.

FIG. 6 shows transfection efficiency and protein expression using aCRISPR vector in U2OS cells (FIG. 6A) and HepG2 cells (FIG. 6B). Thevector contained an OFP reporter gene and was transfected withLIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 reagent into in combinationwith Peptide 1. Bar graphs show relative OFP gene expression (measuredby fluorescence intensity) and fluorescence images below the bar graphsshow quantified fluorescence intensity of corresponding cells expressingOFP.

Genomic cleavage detection: The GENEART® Genomic Cleavage Detection Kitprovides a reliable and rapid method for the detection of locus-specificcleavage. Transfected cells were dissociated with TRYPLE™ Express,washed with Dulbecco's phosphate buffered saline, and pelleted bycentrifugation. Cells were lysed with the Cell Lysis Buffer and ProteinDegrader Mix from the GENEART® Genomic Cleavage Detection Kit. The DNAwas extracted and then PCR-amplified with forward and reverse primers.Denaturing and re-annealing reactions were then performed to randomlyanneal the mutated and un-mutated PCR fragments. Detection enzyme (1 μL)was added, the mix was incubated for 1 hour at 37° C., and then theentire mix was electrophoresed in an E-Gel® EX 2% agarose gel todetermine the percent genome modification. ALPHAVIEW™ Software was usedto determine cleavage efficiency using the following formula: cleavageefficiency=1−[(1−fraction cleaved)^(1/2)].

FIG. 7A shows the cleavage efficiency for TALENs and CRISPRs targetingthe AAVS1 locus in U2OS cells using either LIPOFECTAMINE® 2000 orLIPOFECTAMINE® 3000 in combination with Peptide 1 according to anembodiment.

FIG. 7B shows the cleavage efficiency for TALENs and CRISPRs targetingthe AAVS1 locus in HepG2 cells using either LIPOFECTAMINE® 2000 orLIPOFECTAMINE® 3000 in combination with Peptide 1 according to anembodiment.

Conclusion: U2OS cells, derived from human bone osteosarcoma, and HepG2cells, derived from a human hepatocellular carcinoma, were transfectedwith LIPOFECTAMINE® 3000 and Peptide 1. Both cell lines showed improvedtransfection efficiency and protein expression compared toLIPOFECTAMINE® 2000-mediated transfection. Transfection efficiency andprotein expression were assessed using a CRISPR construct that containsthe OFP reporter gene. U2OS cells transfected with LIPOFECTAMINE® 3000and Peptide 1 had 2-fold improved transfection efficiency (data notshown) and 4-fold improved fluorescence intensity (FIG. 6A). HepG2 cellsshowed 20-fold improvement in transfection efficiency (data not shown)and 80-fold higher fluorescence intensity (FIG. 6B). Significantly,increased TALEN- and CRISPR-mediated cleavage was seen for the AAVS1target locus in both cell lines transfected with LIPOFECTAMINE® 3000 andPeptide 1, demonstrating that increasing the transfection efficiencyand, by implication, protein expression, will increase the cleavage rateof TALENs and CRISPRs. U2OS cells transfected with LIPOFECTAMINE® 3000and Peptide 1 showed 1.5-fold improved TALEN cleavage efficiency andslightly improved CRISPR cleavage (FIG. 7A). HepG2 cells had 3-foldhigher cleavage efficiency for TALENs and 8-fold higher for CRISPRs(FIG. 7B).

Example 8. Lipid Transfection Reagent and Peptide Are Required toEnhance Transfection

HeLa cells were seeded onto 96-well plates and transfected for 48 hrswith 0.2 m/well of pcDNAEF1a/emGFP using either 0.05 μl, 0.1 μl, 0.2 μl,0.3 μl, 0.4 μl, or 0.5 μl of LIPOFECTAMINE® 3000 Reagent alone (LF3K),LIPOFECTAMINE® 2000 (LF2K), Peptide 1 alone (Peptide 1), orLIPOFECTAMINE® 3000 in combination with Peptide 1 as described inExample 1 (LF3K+Peptide 1). Transfection efficiency and proteinexpression as measured by Fluorescence intensity (FL1-H) weredetermined. Results are depicted in FIG. 8.

FIG. 8 shows two bar graphs depicting relative transfection efficiency(upper graph, GFP+ as % of Single Cells Only) or relative GFP expressionlevel per cell (lower graph; Single cells Only Mean FL1-H) of HeLa cellstransfected with a GFP expression vector using the indicated doses (inμl) of LIPOFECTAMINE® 3000 alone (LF3K), LIPOFECTAMINE® 2000 (LF2K), apeptide according to an embodiment (p4) or LIPOFECTAMINE® 3000 incombination with a peptide according to an embodiment (LF3K+peptide).The presence of Peptide 1 in the presence of a cationic lipid aggregateformulation (e.g., LIPOFECTAMINE® 3000) significantly enhancestransfection efficiency and protein expression.

Example 9. Full-Length Peptides are Required for Optimal Transfection

The following peptides were outlined schematically in FIGS. 9A, 10A and11A synthesized and dissolved in ultra-pure water as described inExample 1 for Peptide 1. Peptide A (corresponding to the MPP region ofthe non-naturally occurring peptides of the present invention) andhaving the peptide sequence SRRARRSPRESGKKRKRKR (SEQ ID NO. 1); PeptideB (corresponding to the Linker region of the non-naturally occurringpeptides of the present invention) and having the peptide sequenceGGGSGGGSGGGS (SEQ ID NO. 69); Peptide C (corresponding to the Cationicregion of the non-naturally occurring peptides of the present invention)and having the peptide sequence of CP1 RRRRRRRRRRR (SEQ ID NO. 82);Peptide D (corresponding to the Linker region fused to the Cationicregion of the non-naturally occurring peptides of the present invention)and having the peptide sequence GGGSGGGSGGGSRRRRRRRRRRR (SEQ ID NO.108); and Peptide E, corresponding to Peptide 1 and having the sequenceSRRARRSPRESGKKRKRKRGGGSGGGSGGGSRRRRRRRRRRR (SEQ ID NO. 89).

HepG2, A549 and MDA-MB-231 cells were seeded in 24-well plates andtransfected for 48 hrs with 1 μg of pcDNAEF1a/emGFP using eitherLIPOFECTAMINE® 3000 Reagent in combination with Peptide A, Peptide B,Peptide C, Peptide D, Peptide A and B together, Peptide A and Ctogether, Peptide A and D together, Peptide B and C together or PeptideE or Peptide A, Peptide B, Peptide C, Peptide D, Peptide A and Btogether, Peptide A and C together, Peptide A and D together, Peptide Band C together or Peptide E alone without a lipid transfection reagent.Cells were visualized using fluorescent microscopy and transfectionefficiency as measured by percentage of GFP+cells and protein expressionas measured by mean fluorescence per cell was determined as above.Results are summarized in FIGS. 9B, 9C, 10B, 10C, 11B, and 11C.

FIG. 9B depicts a series of fluorescence images to detect GFP expressionin cultured HepG2 cells transfected with an expression vector encodingGFP using LIPOFECTAMINE® 3000 in the presence of the indicated peptideor combination of peptides (shown in FIG. 9A).

FIG. 9C depicts two bar graphs showing mean fluorescence per cell (uppergraph) and transfection efficiency (% GFP+cells) in HepG2 cellstransfected with an expression vector encoding GFP using LIPOFECTAMINE®3000 in the presence of one of the indicated peptides A-E or theindicated combination of peptides (shown in FIG. 9A).

FIG. 10A is a depiction of a peptide map of various peptides or peptidefragments used in the experiments depicted in FIGS. 10B and 10C in A549cells, in which Peptide A is the MPP Peptide alone, Peptide B is theLinker peptide alone, Peptide C is the Cationic peptide alone, Peptide Dis the Linker peptide fused to the Cationic peptide, and peptide E is afull length peptide having Peptide A fused Peptide D.

FIG. 10B depicts a series of fluorescence images to detect GFPexpression in cultured A549 cells transfected with an expression vectorencoding GFP transfected with LIPOFECTAMINE® 3000 in the presence of theindicated peptide or combination of peptides (shown in FIG. 10A).

FIG. 10C depicts two bar graphs showing mean fluorescence per cell(upper graph) and transfection efficiency (% GFP+cells) in A549 cellstransfected with an expression vector encoding GFP using LIPOFECTAMINE®3000 in the presence of one of the indicated peptides A-E or theindicated combination of peptides (shown in FIG. 10A).

FIG. 11A is a depiction of a peptide map of various peptides or peptidefragments used in the experiments depicted in FIGS. 11B and 11C inMDA-MB-231 cells, in which Peptide A is the MPP Peptide alone, Peptide Bis the Linker peptide alone, Peptide C is the Cationic peptide alone,Peptide D is the Linker peptide fused to the Cationic peptide, andpeptide E is a full length peptide having Peptide A fused Peptide D.

FIG. 11B depicts a series of fluorescence images to detect GFPexpression in cultured MDA-MB-231 cells transfected with an expressionvector encoding GFP transfected with LIPOFECTAMINE® 3000 in the presenceof the indicated peptide or combination of peptides (shown in FIG. 11A).

FIG. 11C depicts two bar graphs showing mean fluorescence per cell(upper graph) and transfection efficiency (% GFP+cells) in MDA-MB-231cells transfected with an expression vector encoding GFP usingLIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A-Eor the indicated combination of peptides (shown in FIG. 11A).

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and can be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. In caseof conflict, the specification herein, including definitions, willcontrol. Citation or identification of any reference in this applicationshall not be construed as an admission that such reference is availableas prior art to the present invention.

What is claimed is:
 1. A transfection complex comprising a lipidaggregate composition comprising at least one cationic lipid, apolypeptide having the structure:A-L-B, orB-L-A; wherein; A is a cationic membrane penetrating peptide sequencecomprising 5 to 50 amino acids, L is a linker peptide sequencecomprising the structure

X_(m)—Y_(n)

_(p) or

Y_(n)—X_(m)

_(p), where each X is independently a neutral amino acid, where each Yis independently a neutral polar amino acid, where m is an integer from3 to 50, where n is an integer from 1 to 40, and where p is an integerfrom 1 to 20; and B is a cationic moiety or a cationic peptide sequencehaving a net positive charge at physiologic pH, and a polyanion cargomolecule complexed with the polypeptide, wherein the cationic lipid, thepolypeptide and the polyanion cargo molecule are present in thetransfection complex in non-covalent association.
 2. The transfectioncomplex according to claim 1, wherein A comprises a peptide sequencethat is at least 75% similar to any one of SEQ ID NO. 1-68 or a variantthereof.
 3. The transfection complex according to claim 2, wherein Acomprises a peptide sequence that is at least 75% similar to any one ofSEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4 SEQ ID NO. 13, SEQ ID NO. 14,SEQ ID NO. 53, SEQ ID NO. 58, SEQ ID NO. 67 and SEQ ID NO. 68 or avariant thereof.
 4. The transfection complex according to claim 1,wherein L comprises a 3 to 50 amino acid sequence, wherein at least 50%of the amino acids in the sequence are neutral.
 5. The transfectioncomplex according to claim 1, wherein L comprises a 3 to 50 amino acidsequence, wherein at least 30% of the amino acids in the sequence areneutral and polar.
 6. The transfection complex according to claim 1,wherein each X is independently glycine, alanine, valine, leucine orisoleucine.
 7. The transfection complex according to claim 1, wherein Xis Gly and Y is Ser.
 8. The transfection complex according to claim 1,wherein m>n.
 9. The transfection complex according to claim 1, wherein mis 3, n is 1, and p is
 3. 10. The transfection complex according toclaim 1, wherein B is a peptide sequence comprising 5-20 positivelycharged amino acids.
 11. The transfection complex according to claim 1,wherein B comprises 5-20 Arg residues.
 12. The transfection complexaccording to claim 1, further comprising a neutral lipid.
 13. Thetransfection complex according to claim 1, wherein the cationic lipid isselected from the group consisting ofN-[I-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamonium chloride (DOTMA),dioleoylphosphatidylcholine (DOPE),1,2-Bis(oleoyloxy)-3-(4′-trimethylammonio) propane(DOTAP),1,2-dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol (DOTB),1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC),dihydroxyl-dimyristylspermine tetrahydrochloride (DHDMS),hydroxyl-dimyristylspermine tetrahydrochloride (HDMD), cholesteryl(4′-trimethylammonio)butanoate(ChoTB), cetyltrimethylammonium bromide(CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DORIE),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),O,O′-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammoniumchloride,spermine conjugated to one or more lipids (for example,5-carboxyspermylglycine dioctadecylamide (DOGS),N,NI,NII,NIII-tetramethyl-N,NI,NII,NIIItetrapalmitylspermine (TM-TPS)and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde (DPPES)),lipopolylysine (polylysine conjugated to DOPE),TRIS(Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fattyacids (TF As) and/orpeptides such as trilysyl-alanyl-TRIS mono-, di-,and tri-palmitate, (3β-[N—(N′,N′dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), N-(a-trimethylammonioacetyl)didodecyl-D-glutamatechloride (TMAG), dimethyl dioctadecylammonium bromide(DDAB),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamininiumtrifluoroacetate(DOSPA)and combinations thereof.
 14. The transfection complex according toclaim 1, wherein said cargo molecule is a nucleic acid, optionallywherein said cargo molecule is a DNA or an RNA molecule.
 15. A methodfor delivering a polyanion into a cell, said method comprising: forminga transfection complex comprising one or more cationic lipids, apolyanion cargo molecule, and a polypeptide having the structure:A-L-B or B-L-A; wherein A is a membrane penetrating peptide sequencecomprising 5 to 50 amino acids, L is a linker peptide sequencecomprising the structure

X_(m)—Y_(n)

_(p) or

Y_(n)—X_(m)

_(p), where each X is independently a neutral amino acid, where each Yis independently a neutral polar amino acid, where m is an integer from3 to 50, where n is an integer from 1 to 40, and where p is an integerfrom 1 to 20; and B is a cationic moiety or a cationic peptide sequencehaving a net positive charge at physiologic pH; and contacting a cellwith the transfection complex, wherein the one or more cationic lipids,the polypeptide and the polyanion cargo molecule are present in thetransfection complex in non-covalent association.
 16. The methodaccording to claim 15, wherein A comprises a peptide sequence that is atleast 75% similar to any one of SEQ ID NO. 1-68 or a variant thereof.17. The method according to claim 15, wherein forming the transfectioncomplex comprises contacting the polyanion cargo molecule with thepolypeptide prior to addition of the one or more cationic lipids. 18.The method according to claim 15, wherein the transfection complexfurther comprises one or more neutral lipids.
 19. The method accordingto claim 18, wherein forming the transfection complex comprises (i)forming a lipid aggregate comprising the one or more cationic lipids andthe one or more neutral lipids and (ii) contacting the lipid aggregatewith the polyanion cargo molecule and the polypeptide.
 20. The methodaccording to claim 15, wherein the one or more cationic lipids isselected from the group consisting ofN-[I-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamonium chloride (DOTMA),dioleoylphosphatidylcholine (DOPE),1,2-Bis(oleoyloxy)-3-(4′-trimethylammonio) propane(DOTAP),1,2-dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol (DOTB),1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC),dihydroxyl-dimyristylspermine tetrahydrochloride (DHDMS),hydroxyl-dimyristylspermine tetrahydrochloride (HDMD), cholesteryl(4′-trimethylammonio) butanoate(ChoTB), cetyltrimethylammonium bromide(CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DORIE),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DMRIE),O,O′-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammoniumchloride,spermine conjugated to one or more lipids (for example,5-carboxyspermylglycine dioctadecylamide (DOGS),N,NI,NII,NIII-tetramethyl-N,NI,NII,NIIItetrapalmitylspermine (TM-TPS)and dipalmitoylphasphatidylethanolamine 5-carboxyspermylaminde (DPPES)),lipopolylysine (polylysine conjugated to DOPE),TRIS(Tris(hydroxymethyl)aminomethane, tromethamine) conjugated fattyacids (TF As) and/orpeptides such as trilysyl-alanyl-TRIS mono-, di-,and tri-palmitate, (3β-[N—(N′,N′dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), N-(a-trimethylammonioacetyl)didodecyl-D-glutamatechloride (TMAG), dimethyl dioctadecylammonium bromide(DDAB),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamininiumtrifluoroacetate(DOSPA)and combinations thereof.